Chiral Cyclic Beta-Amino Acids and their Derivatives, Pharmaceutical Compositions Containing Them and the Use of Such Compounds

ABSTRACT

The invention relates to chiral cyclic β-amino acids of Formula (I) and their salts formed with pharmaceutically acceptable acids or bases, wherein the main meanings of the substituents are as follows: R stands for C 1-4  Alk; X stands for —COOH, —CONH 2 , —CONH(C 1-4  Alk), —CON(C 1-4  Alk) 2 , —COO(C 1-4  Alk), —COPhe-O-(C 1-4  Alk) or —CH 2 OH; Y stands for —NH 2 , —NHBoc, —NHFmoc, —NH(C 1-4  Alk), —N(C 1-4  Alk) 2 , —NHCH 2 Ph, or Ar—NH—C(=X 0 )—N(R 0 )— wherein Ar stands for a phenyl group substituted by C 1-4  alkoxy or halogen, X 0  stands for O or S, and R 0  stands for hydrogen or benzyl; and X+Y stands for —CONH— vagy —CON(Boc)-; with the proviso that when X stands for —COOH, then Y may be only different from —NH 2 . The invention also relates to pharmaceutical compositions having multidrug-resistance reversing effect that contain one or more compound(s) of Formula (I) or a salt thereof and inert pharmaceutical carriers and/or auxiliary agents. The invention also relates to carboxylic acids of Formula (XX) and their salts.

FIELD OF THE INVENTION

The invention relates to chiral cyclic β-amino acids and theirderivatives, pharmaceutical compositions containing them and the use ofsuch compounds.

BACKGROUND OF THE INVENTION

The systematic investigations concerning stereoisomeric alicyclicβ-amino acids [F. Fülöp Chem. Rev. 101, 2181-2204 (2001)] wereconsidered for a long time as having only theoretical interest but aboutin the last decade it has turned out that several representatives ofthese compounds can be found in nature and have per se remarkablepharmacological effects and, resp., they are building elements ofbiologically active more complex compounds. The alicyclic β-amino acidsplay an important role even in the preparation of modified(“non-natural”) analogues of biologically active peptids, and bychanging the α-amino acids to β-amino acids it is possible to modify theeffect of the peptide and/or its stability can be increased.

Due to the occurrence in nature of the alicyclic β-amino acids and suchacids of other types as well as the valuable biological activity oftheir derivatives and their manifold synthetic useability, several novelenantioselective processes have been elaborated for their synthesis [F.Fülöp, Chem. Rev. 101, 2181-2204 (2001); F. Fülöp, T. A. Martinek and G.K. Tóth, Chem. Soc. Rev. 35, 323-334 (2006)]. This can be followed inthe preparation of β-amino acid enantiomers and their use of everincreasing degree in asymmetric reactions.

A novel direction in the preparation of chiral β-amino acids is theβ-amino acid synthesis from chiral monoterpene derivatives [e.g. from(+)- and (−)-α-pinene, (+)-3-carene] [Z. Szakonyi, T. Martinek, A.Hetényi and F. Fülöp, Tetrahedron:Asymmetry, 11, 4571-4579 (2000); S.Gyónfalvi, Z. Szakonyi and F. Fülöp: Tetrahedron:Asymmentry, 14,3965-3972 (2003)]. From the thus-obtained β-amino acid derivativeshaving monoterpene skeleton 1,3-heterocyclics of varied structures havebeen prepared [Z. Szakonyi, T. Martinek, A. Hetényi and F. Fülöp,Tetrahedron:Asymmetry, 11 4571-4579 (2000); S. Gyönfalvi, Z. Szakonyiand F. Fülöp, Tetrahedron:Asymmetry, 14, 3965-3972 (2003); Z. Szakonyiand F. Fülöp, Arkivoc, xiv, 225-232 (2003)].

It has been observed, however, that, due to the monoterpene skeletonwherein the amino group of the amino acid is attached to a tertiarycarbon atom, the reactivity of the compounds in relation to thereactivity of the simpler alicyclic analogues is decreased in a highdegree. However, by the suitable choice of the starting materials theamino group can be attached to the secondary carbon atom whereby theβ-amino acid derivatives get normal reactivity, simultaneouslypreserving the enantiomeric purity secured by the natural monoterpenestarting materials.

It is known that most of the cancer patients have to be treated withchemotherapeutical agents, too. However, the effectivity of thechemotherapy is strongly decreased by the appearance of resistanceagainst cytostatica, especially the multidrug resistance (MDR) againstchemotherapeutical drugs. The resistance against the anti-cancerousdrugs is often transmitted by the expression of excessive degree of themembrane pump called P-glycoprotein (P-gp or MDR1), and this protein iscoded by a gene called mdr1.

The P-gp is a member of the ABC superfamily. These transportersdepending on ATP decrease the intracellular drug concentration below thecytotoxic levels, whereby the cytostatica become increasingly lesseffective and the toxic side-effects become increasingly stronger duringthe treatment, since the membrane pump transports the cytostatica fromthe intracellular space to the extracellular space. The strong decreaseof the intracellular cytostaticum concentration during the treatmentrenders possible the survival and metastasis of the pathogenic tumourcells [E. Andicott et al., Ann. Rev. Bioch. 58, 137-171 (1989)].

Consequently, the surmounting of the multidrug resistance is extremelyimportant since in this way numerous cancers and contagious illnessescan be successfully treated. Today no effective drug is known by the aidof which this aim could be attained in vivo.

The investigation of multidrug resistance gains ground in anincreasingly wide sphere. A very promising method of surmounting MDR isto develop MDR-modulators which can inhibit the activity of theP-glycoprotein.

SUMMARY OF THE INVENTION

Surprisingly it has been found that the novel terpene derivativesaccording to the invention are biologically active, non-toxic compoundswhich can surmount in a very low concentration the resistance againstchemotherapeutical drugs of the pathological tumour) cells and inhibitthe pump activity of the pathological MDR tumour cells.

Based on the above, the invention relates to chiral2-amino-6,6-dimethyl-bicyclo[3.1.1]heptane-3-carboxylic acid derivativesof general formula (I)—

wherein

R stands for C₁₋₄ Alk;

X stands for —COOH, —CONH₂, —CONH(C₁₋₄ Alk), —CON(C₁₋₄ Alk)₂, —COO(C₁₋₄Alk), —COPhe-O-(C₁₋₄ Alk) or —CH₂OH;

Y stands for —NH₂, —NHBoc, —NHFmoc, —NH(C₁₋₄ Alk), —N(C₁₋₄ Alk)₂,—NHCH₂Ph, or Ar—NH—C(=X⁰)—N(R⁰)— wherein Ar stands for a phenyl groupsubstituted by one or two C₁₋₄ alkoxy group(s) or by one halogen, X⁰stands for O or S, and R⁰ stands for hydrogen or benzyl; and

X+Y stands for —CONH— vagy —CON(Boc)-;

with the proviso that when X stands for —COON, then Y may be onlydifferent from —NH₂— and to their salts formed with pharmaceuticallyacceptable acids or bases.

In the formulas Boc stands for tert-butoxycarbonyl, Alk stands foralkyl, Hlg stands for halogen, Fmoc stands forfluorenyl-methoxycarbonyl, Phe stands for phenylalanine and Me standsfor methyl.

It is very surprising that the compounds according to the inventioninduce the chemical sensitisation of the resistant Mdr-tumour cells andsuppress their efflux-pump activity. That results in so high values ofthe intracellular chemotherapeutics concentration that no difference canbe observed in relation to the parenteral cells.

Consequently, the compounds according to the invention are biologicallyactive and suppress the efflux-pump activity of malignant MDR-tumourcells even at very low concentrations. They are not toxic in vivo evenat higher concentrations. They are active immediately after the contactwith the cells. Thus, the compounds according to the invention enhancein the first line the chemotherapeutic efficiency, that is, they can beused in the chemotherapy with other active agents too.

DETAILED DESCRIPTION OF THE INVENTION

The following compounds according to the invention have shown especiallyadvantageous effects:

(1R,2R,5S,7R)-N-tert-butoxycarbonyl-8,8-dimethyl-3-azatricyclo-[5.1.1.0^(2,5)]nonane-4-one(compound of formula 11),

(1R,2R,3R,5R)-2-(9H-fluoren-9-yl-methoxycarbonylamino)-6,6-dimethyl-bicyclo[3.1.1]heptane-3-carboxylicacid (compound of formula 30),

(1R,2R,3S,5R)-1-(3-ethoxycarbonyl-6,6-dimethyl-bicyclo-[3.1.1]heptane-2-yl)-3-(3-methoxyphenyl)-thiourea(compound of formula 44),

(1R,2R,3S,5R)-1-(3-ethoxycarbonyl-6,6-dimethyl-bicyclo[3.1.1]heptane-2-yl)-3-(3,4-dimethoxy-phenylethyl)-thiourea(compound of formula 46),

(1R,2R,3S,5R)-1-benzyl-1-(3-ethoxycarbonyl-6,6-dimethyl-bicyclo[3.1.1]heptane-2-yl)-3-(3-methoxyphenyl)-thiourea(compound of formula 47),

(1R,2R,3R,5R)-1-(3-ethoxycarbonyl-6,6-dimethyl-bicyclo[3.1.1]heptane-2-yl)-3(3-methoxyphenyl)-thiourea(compound of formula 49),

(1R,2R,3R,5R)-1-(3-ethoxycarbonyl-6,6-dimethyl-bicyclo[3.1.1]heptane-2-yl)-3-(4-chlorophenyl)-urea(compound of formula 50),

(1R,2R,3R,5R)-1-(3-ethoxycarbonyl-6,6-dimethyl-bicyclo[3.1.1]heptane-2-yl)-3-(3,4-dimethoxyphenyl-ethyl)-thiourea(compound of formula 51),

(1R,2R,3R,5R)-1-benzyl-1-(3-ethoxycarbonyl-6,6-dimethyl-bicyclo[3.1.1]heptane-2-yl)-3-(3-methoxyphenyl)-thiourea(compound of formula 52), and

(1S,2S,3S,5S)-1-(3-ethoxycarbonil-6,6-dimethyl-bicyclo[3.1.1]heptane-2-yl)-3-(4-chlorophenyl)-urea(compound of formula 56).

The invention also relates to the preparation of the compounds ofgeneral formula (I). The process according to the invention can berealized according to the enclosed reaction scheme 1 wherein themeanings of the symbols and abbreviations, resp., are as follows:

R¹=alkyl

R²=alkyl or aralkyl

R³=alkyl

R⁴=alkyl, aralkyl or aryl

R⁵=alkyl

R⁶=H, alkyl, aralkyl or aryl

P=N-protecting group (e.g. Boc, Fmoc, etc.)

X¹=activating group (e.g. halogen, acid residue)

Y¹=O or S

CSI=chlorosulphonyl isocyanate

LAH=lithium aluminium hydride

TEA=triethylamine

THF=tetrahydrofuran.

Based on reaction scheme 1, for preparing the compounds according to theinvention one proceeds as follows:

β-lactam enantiomers of formula (III) are prepared by reacting suitablemonoterpenes with chlorosulphonyl isocyanate and then treating theintermediary product with an alkali;

the amino group of the compounds of formula (III) is preferablyprotected with an alkoxycarbonyl group, then N-protected acid amides offormula (V) are prepared by aminolysis of the β-lactam ring of thethus-obtained compound of formula (IV);

by carrying out the acid solvolysis of the β-lactams of formula (III)with inorganic acids in water cis-amino acids of formula (VIa/cis),whereas by carrying out the solvolysis in suitable alcohols (R₃OH)esters of formula (VIIa/cis) are directly prepared;

trans-amino acid esters of formula (VIIa/trans) are prepared by thealkaline isomerisation of cis-amino acid esters of formula (VIIa/cis),and by the hydrolysis of the compounds of formula (VIIa/trans) thecorresponding trans-amino acids of formula (VIa/trans) are prepared;

the N-substituted amino acid esters of formula (VIIIb) are prepared bythe reductive alkylation of amino acid esters of formula (VIIa), and byhydrolysis of the thus-obtained compounds the correspondingN-substituted amino acids of formula (VIIb) are prepared;

the amino group of the amino acids of formula (VIa) or (VIb) isprotected preferably with an alkoxycarbonyl group, and the thus-obtainedN-protected amino acids of formula (VIII) are coupled preferably by themixed anhydride method with other amino acids and amino acid esters,resp., whereby dipeptides of general formula (IX) are prepared;

by reducing the amino acid esters of formula (VIIa) or (VIIb) preferablywith inorganic hydrides amino alcohols of formula (XII) are prepared,and by carrying out the reduction with alkyl- or aryl-isocyanates and,resp., -isothiocyanates urea and, resp., thiourea derivatives of formula(XI) are prepared.

The compounds of formulas (III) and (IV) exist only in 1,2-cisdiastereomeric form, and the formulas relate to both enantiomers,whereas the compounds of formulas (V) to (XII) also exist in 1,2-cis and1,2-trans diastereomeric forms and the formulas can relate to bothenantiomers. To compound series cis can be prepared directly from theenantiomers of formula (III), while the trans series can be obtained bythe alkaline epimerisation of the cis-amino acid ester of formula (VIIa)(see Example 23).

The concrete methods for preparing the compounds according to theinvention are described, based on reaction schemes 3-12, in Examples1-47.

The reactions can be followed by thin-layer chromatography. Theprocessing up can be carried out by evaporation or extraction. Theproduct can be purified by crystallization or chromatography. Thestructure and purity of the compounds can be proved and checked, resp.,by NMR spectroscopy, measuring the melting point and/or CHNmicroanalysis.

The invention also relates to multidrug resistance reversingpharmaceutical compositions containing compounds of general formula (I)or their salts. In compliance with the invention these compositions canbe prepared as follows: one or more compound(s) of general formula (I)or the salts thereof is/are mixed with usual inert pharmaceuticalcarriers and/or auxiliary agents, whereby MDR-reversing pharmaceuticalcompositions are obtained.

Furthermore, the invention also relates to the use of the compounds ofgeneral formula (I) and/or the salts of such compounds for preparingpharmaceutical compositions reversing multidrug resistance.

The scope of the invention also relates to the novel2-amino-6,6-dimethyl-bicyclo[3.1.1]heptane-3-carboxylic acids of generalformula (XX) and their salts formed with pharmaceutically acceptableacids and bases, resp., obtained as intermediary products when preparingthe novel compounds of general formula (I). The compounds of formulas 6,7, 8, and 9 belong to the scope of the compounds of general formula(XX); their preparation is described in Examples 5, 10, 26.b), and 28,resp.

The compounds according to the invention can be transformed withphysiologically acceptable acids and bases, resp., to salts. Inorganicor organic acids of this kind are the hydrochloric acid, hydrobromicacid, phosphoric acid, formic acid, acetic acid, propionic acid,glycolic acid, lactic acid, citric acid, maleic acid, fumaric acid,anthranilic acid, methansulphonic acid, naphthalenesulfonic acid,sulfanilic acid, and cinnamic acid.

Sodium hydroxide, potassium hydroxide, and calcium hydroxide arementioned as specifically acceptable bases.

In the practice the compounds according to the invention of generalformula (I) can be administered in solid, liquid or spray form alone ortogether with one or more usual pharmaceutical carrier(s) or auxiliaryagent(s). Accordingly, the invention also relates to pharmaceuticalcompositions that contain an effective amount of the compounds ofgeneral formula (I) together with a pharmaceutically acceptable carrier.As a matter of course, the compounds according to the invention can alsobe administered together with other MDR-reversing compounds.

As solid carriers that can be used in the compositions according to theinvention one or more material(s) can be used which simultaneously serveas sliding agent, solubilizing agent, suspending agent, binder, filler,pressing agent, disintegrating agent, flavouring agent or capsulatingagents.

In the case of powders the carrier can be a finely divided solidmaterial that is mixed with finely divided particles of the compounds ofgeneral formula (I). In the case of tablets, the compound of generalformula (I) is mixed in a suitable ratio with a carrier which has therequired pressing properties, and the mixture is pressed to the desiredform and size.

The above-mentioned powders and tablets contain the compound of generalformula (I) in an amount ranging up to 99% by mass.

The compositions of spray form are generally used as aerosolcompositions for absorption on the skin surface or through the lungs.

As examples of the solid carriers which can be used in the compositionsaccording to the invention the following are mentioned by way ofexample: talc, calcium phosphate, magnesium stearate, lactose, dextrin,starch, gelatine, cellulose, methyl cellulose, sodiumcarboxymethylcellulose, polyvinyl pyrrolidone and waxes of low meltingpoint.

In the compositions according to the invention any pharmaceuticallyacceptable liquid carrier can be used which is suitable for preparingsolutions, suspensions, emulsions, syrups and therapeutic drinks. Thecompounds of formula (I) can be dissolved or suspended in apharmaceutically acceptable liquid carrier such as water, organicsolvents or pharmaceutically acceptable oils or fats or a mixturethereof. The liquid compositions may contain other suitablepharmaceutical additives such as solubilising agents, emulgators,buffers, conserving agents, sweeteners, flavouring agents, suspendingagents, thickeners, colouring agents, viscosity regulators, stabilizers,osmotic pressure regulators, and similar agents.

For oral or parenteral administration the following examples of liquidcarriers are mentioned: water, aqueous solutions containing additivessuch as cellulose derivatives, preferably sodium carboxymethylcellulose,alcohols including primary and secondary alcohols such as glycols,alcohol derivatives or oils such as fractionated coconut oil and arachisoil. In case of parenteral administration the carrier oil can be anester such as ethyl oleate or isopropyl myristate.

The orally administrable compositions according to the invention can beliquid or solid.

The compositions according to the invention that are sterile solutionsor suspensions can be used as intramuscular, intraperitoneal orsubcutaneous injections. The sterile solutions can be administeredintravenous too.

The compounds according to the invention can be formulated together withpharmaceutically effective other materials. The selection of suchmaterials depends on the aim of using the compositions.

In the compositions the concentration of the compounds of generalformula (I) depends on several factors such as the method ofadministration, the chemical nature of the compounds, the state andclinical indication of the patient. Therefore, the concentration canvary in a wide range. Generally the concentration of the active agentcan be 0.002-99% by mass, e.g. 0.01-70% by mass, preferably 0.05-40% bymass, more preferably 0.1-20% by mass.

The compositions according to the invention can be administeredenterally, parenterally, locally, rectally or systemicly depending onthe prescription and the active agent used, e. g. in the form oftablets, capsules, powder, granules, syrup, spray, or injectionsolution.

The compositions for enteral administration can be e. g. powders, simpleor coated tablets, tablets with protracted effect, soft capsules, hardcapsules, rectal suppositories, suspensions and solutions which, ifdesired, may contain the active agents together with one or more usualcarriers.

The compositions for parenteral administration can be e.g. preparationsfor intradermal, subcutaneous, intraperitoneal or intravenous injectionsor infusions. Other parenteral compositions can be applied not only onthe skin but also on the mucous membrane. Such local compositions can bee.g. gels, creams, ointments, shampoos, soaps, sprays, rinsing agents,smears, aerosols, and other pharmaceutical preparations suitable forlocal administration.

The compounds according the inventions preserve their effect when usedin any form, e.g. when used in the usual oral or rectal or injectionform.

In the case of oral administration the daily dose of the compoundsaccording to the invention amounts in adults to 200-1000 mg, preferably50-500 mg, more preferably 20-100 mg. These doses can be increased ordecreased depending on the state of the patients.

The toxic effect of the compounds was measured on MRC-5 human fibroblastcell lines in accordance with the method that has been described in thearticle published by Molnár et al. [L. Molnár, G. M. Keserü, Á. Papp, Z.Lörincz, G. Ambrus és F. Darvas.: A neural network based classificationscheme for cytotoxicity predictions: Validation on 30,000 compounds,Bioorg. Med. Chem. Lett. 16(4), 1037-9 (2006)]. The cells were incubatedfor a cell cycle (24 hours) with solutions of various concentrationsprepared from the compounds, in the presence of 1% of DMSO. 3 parallelmeasurements were carried out with 6 different concentrations. The ratioof the viable cells was determined by reducing the Alamaar Blue(resazurine) reagent that was followed fluorimetrically. The measuredmaterials were not toxic on the given cell line in a concentration of128 μmol after incubation for one day.

In the following Table 1 the cytotoxicity of some compounds according tothe invention is given.

TABLE 1 Number of the Survival (%) compound formula [c = 128 μM] 38125.3 44 128.1 45 176.7 46 182.2 47 97.7 49 250.9 50 167.6 52 113.0 55214.1 56 116.8

The preparation of the starting compounds (1S,5S)- and (1R,5R)-apopinene(2 and 5) used for the synthesis of the compounds according to theinvention was carried out in compliance with the literature method, inthe way as represented on the enclosed reaction scheme 2 [Shibuya K.,Synth. Commun., 24, 2923-2941 (1994); Lightner D. A.; Crist, B. V.Tetrahedron, 41, 3021-3028 (1985)]. The (−)-(1R,5S)-myrtenale (1) andthe (+)-(1R,5R)-α-pinene (3) are commercially available products(Sigma-Aldrich Kft.).

Reaction schemes 3-12 show the synthesis of the compounds according tothe invention. The detailed description of the experiments and thephysical and chemical characteristics of the compounds prepared aregiven in the individual Examples.

Schemes 2 to 12 are illustrated in FIG. 1 in the enclosed drawing.

Experimental Section

General Procedures. ¹H NMR spectra were recorded on a Bruker Avance DRX400 spectrometer [400.13 MHz (¹H) and 100.61 MHz (¹³C), δ=0 (TMS)] inCDCl₃ or in D₂O in a 5 mm tube. Chemical shifts are expressed in ppm (δ)relative to TMS as internal reference. J values are given in Hz.

The enantiomer purity of the reveal compounds were performed by GCmeasurements on a Chrompack CP-9002 system, consisting of a 901A FlameIonization Detector and a Maestro II Chromatography data system(Chrompack International B.V., Middelburg, The Netherlands). The columnused for direct separation of the enantiomers was a CHIRASIL-DEX CBcolumn (2500×0.25 mm I.D.) at 170° C., 80 kPa for azetidinones and 100°C., 25 kPa for apopinene. Optical rotations were obtained with aPerkin-Elmer 341 polarimeter. FT-IR spectra were recorded on aPerkin-Elmer model 1000 spectrophotometer. Microanalyses were determinedon a Perkin-Elmer 2400 elemental analyser. Melting points weredetermined on a Kofler apparatus and are uncorrected.

EXAMPLE 1(1R,2R,5S,7R)-8,8-Dimethyl-3-azatricyclo[5.1.1.0^(2,5)]nonan-4-one(compound 10) (Scheme 3)

A mixture of 12.21 g (100.0 mmol) of (−)-(1S,5S)-apopinene (2), preparedvia literature method [Lightner, D. A.; Crist, B. V. Tetrahedron, 41,3021-3028 (1985)], and 14.30 g (101.2 mmol) of chlorosulfonyl isocyanate(CSI) was stirred in 300 ml of dry diethyl ether for 48 h at roomtemperature. 20.4 g (162 mmol) of dry sodium sulfite in 140 ml water wasthen cautiously added dropwise to the solution. The pH was held at 7-8by the addition of 20% aqueous potassium hydroxide. After 2 h stirringat the appropriate pH, the organic phase was separated and the aqueouslayer was extracted with diethyl ether (2×100 ml). The combined organiclayer was dried (Na₂SO₄) and evaporated, and the white crystallineproduct obtained was recrystallized from n-hexane.

Isolated compound: 13.54 g (82%); mp: 68-72° C.; [α]_(D) ²⁰=−80.0(c=0.5, MeOH; ee >99%); IR=3247, 2914, 1710, 1380, 1256, 1189 cm⁻¹.Anal. Calcd. for C₁₀H₁₅NO (165.23): C, 72.69; H, 9.15; N, 8.48; Found:C, 72.52; H, 9.27; N, 8.31. ¹H NMR (CDCl₃) δ (ppm): 0.88 (3H, s, Me-8),1.30 (3H, s, Me-8), 1.50 (1H, d, H-9, J=11.1 Hz), 1.82-1.98 (2H, m),2.07-2.29 (3H, m), 3.28 (1H, dd, H-5, J=10.32 Hz), 3.95-4.00 (1H, m),5.87 (1H, s, NH); ¹³C NMR (CDCl₃) δ (ppm): 19.7 (Me-6), 23.3 (CH₂), 24.7(CH₂), 26.8 (Me-6), 40.0 (C_(q)), 41.9 (CH), 43.7 (CH), 44.9 (CHC=O),51.8 (CHN), 173.9 (C═O).

EXAMPLE 2(1S,2S,5R,7S)-8,8-Dimethyl-3-azatricyclo[5.1.1.0^(2,5)]nonan-4-one(compound 33) (Scheme 7)

The synthesis of 1S,2S,5R,7S enantiomer 33 was accomplished by analogywith Example 1, starting from (+)-(1R,5R)-apopinene 5 which was preparedvia literature method [Lightner, D. A.; Crist, B. V. Tetrahedron, 41,3021-3028 (1985)]; [α]_(D) ²⁰=+61.5 (c=0.5, MeOH; ee=90%); all thespectroscopic data and mp were similar to those for (−)-enantiomer 10.

EXAMPLE 3(1R,2R,5S,7R)-N-tert-Butoxycarbonyl-8,8-dimethyl-3-azatricyclo[5.1.1.0^(2,5)]-nonan-4-one(compound 11) (Scheme 3)

To a stirred solution of 0.30 g (1.8 mmol) of(1R,2R,5S,7R)-8,8-dimethyl-3-azatricyclo[5.1.1.0^(2,5)]nonan-4-one (10)prepared according to Example 1, and dry THF (10 ml), triethylamine(0.47 g, 4.6 mmol), di-tert-butyl dicarbonate (0.51 g, 2.3 mmol) and acatalytic amount of 4-dimethylaminopyridine were added while stirring.After stirring for 6 h at room temperature (the reaction was monitoredby means of TLC), the mixture was evaporated to dryness. The oilyresidue obtained was purified by flash chromatography on a silica gelcolumn (n-hexane:ethyl acetate=9:1), resulting in a white crystallineproduct (0.43 g, 89%): mp: 64-66° C.; [α]_(D) ²⁰=−41.1 (c=0.5, MeOH);IR=2926, 1803, 1707, 1349, 1156 cm⁻¹. Anal. Calcd. for C₁₅H₂₃NO₃(265.35): C, 67.90; H, 8.74; N, 5.28. Found: C, 68.16; H, 8.54; N, 5.35.¹H NMR (CDCl₃) δ (ppm): 0.89 (3H, s), 1.31 0.89 (3H, s), 1.35 (1H, d,J=11.6 Hz, H-9), 1.50 (9H, s), 1.81-2.00 (2H, m, H-6, H-7), 2.14-2.25(2H, m, H-6, H-9), 2.51-2.57 (1H, m, H-1), 3.29 (1H, dd, J=10.3, 6.2 Hz,H-2), 4.27 (1H, dd, J=5.6, 4.0 Hz, H-5). ¹³C NMR (CDCl₃) δ (ppm): 20.5(Me), 24.3 (C-9), 25.5 (C-6), 27.3 (Me), 28.7 (Me₃C), 39.8 (C-8), 42.1,42.5, 44.1, 55.5, 83.4 (Me₃C), 148.6 (C-4), 170.5 (Me₃COC=O).

EXAMPLE 4 tert-Butyl(1R,2R,3S,5R)-(2-methylaminocarbonyl-6,6-dimethylbicyclo[3.1.1]-hept-3-yl)carbamate(compound 12) (Scheme 3)

0.29 g (1.08 mmol) of the (1R,2R,5S,7R) N-Boc β-lactam (11), preparedaccording to Example 3, was dissolved in a 25% solution of methylaminein dry methanol (50 ml). The reaction mixture was allowed to stand at 4°C. for 12 h. After evaporation to dryness, the yellow crude product waspurified by flash chromatography on a silica gel column (n-hexane:ethylacetate=4:1) to give 12 as white crystals.

Isolated compound: 0.28 g (86%): mp: 104-105° C.; [α]_(D) ²⁰=+52.7(c=0.5, MeOH); IR=3330, 2979, 1668, 1539, 1179. Anal. Calcd. forC₁₆H₂₈N₂O₃ (296.41): C, 64.83; H, 9.52; N, 9.45. Found: C, 64.97; H,10.05; N, 9.23. ¹H NMR (CDCl₃) δ (ppm): 0.89 (3H, s, Me-6), 1.01 (3H, s,Me-7), 1.22 (3H, s, Me-6), 1.41 (9H, s), 1.31 (1H, d, J=10.1 HZ),1.74-1.96 (3H, m), 2.12-2.28 (2H, m), 2.77 (3H, d, J=5.0 Hz, NHMe), 3.02(1H, dt, J=10.1, 3.5 Hz, H-3), 4.33 (1H, t, J=9.7 Hz, H-2), 5.04 (1H, d,J=9.7 Hz, NHMe), 5.73 (1H, br s, NHBoc). ¹³C NMR (CDCl₃) δ (ppm): 21.0(Me), 25.4 (CH₂), 26.8 (Me), 27.3 (CH), 28.8 (CH₂), 29.0 (3×Me), 39.7(C_(q)), 40.0 (CH), 41.0 (CH), 46.8 (CH), 49.8 (CH), 79.8 (CMe₃), 156.6,176.1.

EXAMPLE 5(1R,2R,3S,5R)-2-Amino-6,6-dimethylbicyclo[3.1.1]heptan-3-carboxylic acid(compound 13) (Scheme 3)

0.50 g (3.0 mmol) of(1R,2R,5S,7R)-8,8-dimethyl-3-azatricyclo[5.1.1.0^(2,5)]nonan-4-one (10),prepared according to Example 1, was stirred in a solution of 5 ml of15% hydrochloric acid at room temperature. When the mixture became clear(approx. 1 h), the solution was evaporated to dryness and the resultedwhite crystalline product was washed with acetone and filtered off. 0.61g (94%) of(1R,2R,3S,5R)-2-amino-6,6-dimethyl-bicyclo[3.1.1]heptan-3-carboxylicacid hydrochloride (6) was isolated by this method; mp: 248-249° C.;[α]_(D) ²⁰+22.5 (c=0.5, MeOH; ee >99%); IR=2904, 1724, 1584, 1489, 1175cm⁻¹. Anal. Calcd. for C₁₀H₁₈ClNO₂ (219.71): C, 54.67; H, 8.26; N, 6.38.Found: C, 54.87; H, 8.02; N, 6.71. ¹H NMR (D₂O) δ (ppm): 0.89 (3H, s,Me-6), 1.27 (3H, s, Me-6), 1.48 (1H, d, J=11.1 Hz), 2.04-2.16 (2H, m),2.31-2.39 (2H, m) 3.39 (1H, dt, J=10.7, 3.6 Hz), 3.98 (1H, d, J=9.6 Hz).¹³C NMR (CDCl₃) δ (ppm): 19.7 (Me), 24.3 (CH₂), 25.8 (Me), 29.4 (CH₂),34.4 (CH), 39.3 (C_(q)), 39.6 (CH), 44.2 (CH), 50.2 (CH), 178.5 (C═O).

The (1R,2R,3S,5R)-2-amino-6,6-dimethylbicyclo[3.1.1]heptan-3-carboxylicacid hydrochloride (6) prepared above, was dissolved in 5 ml of waterand the pH of the solution was adjusted to pH=7.4 with 10% solution ofNaHCO₃ under ice cooling in the presence of methylthymol blue indicator.After 1 h stirring at 0° C. the precipitated product 13 was filtered andwashed with a small amount of ice-cold distilled water.

Isolated compound: 0.29 g (58%); mp: 270° C.; [α]_(D) ²⁰=−1.6 (c=0.50,MeOH); IR=3243, 2972, 1625, 1576, 1474, 1382 cm⁻¹. Anal. Calcd. forC₁₀H₁₇NO₂ (183.25): C, 65.54; H, 9.35; N, 7.64. Found: C, 65.48; H,9.57; N, 7.42. ¹H NMR (CD₃OD) δ (ppm): 0.99 (3H, s), 1.36 (3H, s), 1.73(1H, d, H-4, J=10.6 Hz), 2.00-2.06 (1H, m), 2.12 (1H, dt, J=2.0, 5.5Hz), 2.26-2.39 (3H, m), 3.02 (1H, dt, J=9.6, 4.5 Hz), 3.82 (1H, dt,J=9.6, 2.0 Hz). ¹³C NMR (CDCl₃) δ (ppm): 22.9 (Me), 26.1 (CH₂), 27.6(Me), 32.5 (CH₂), 37.8 (CH), 39.3 (C_(q)), 42.3 (CH), 47.2 (CH), 52.0(CH), 183.1 (C═O).

EXAMPLE 6 Methyl(1R,2R,3S,5R)-2-amino-6,6-dimethylbicyclo[3.1.1]heptan-3-carboxylatehydrochloride (compound 14) (Scheme 3)

Compound 14 was prepared by two methods:

Method 6a

2.0 g (12.1 mmol) of(1R,2R,5S,7R)-8,8-dimethyl-3-azatricyclo[5.1.1.0^(2,5)]nonan-4-one (10),prepared according to Example 1, was stirred in solution of 10%hydrochloric acid in dry methanol (20 ml) at room temperature. After 1.5h stirring, the solution was evaporated to dryness and the resultedcrystalline product was recrystallized from isopropyl ether/ethylacetate mixture.

Isolated compound: 1.98 g (71%); mp: 157-160° C.; [α]_(D) ²⁰=+4.8(c=0.5, MeOH); IR=2926, 1724, 1498, 1205 cm⁻¹. Anal. Calcd. forC₁₁H₂₀ClNO₂ (233.74): C, 56.52; H, 8.62; N, 5.99. Found: C, 56.73; H,9.01; N, 6.17. ¹H NMR (CDCl₃) δ (ppm): 0.97 (3H, s, Me-6), 1.35 (3H, s,Me-6), 1.54 (1H, d, H-4, J=11.1 Hz), 2.08-2.22 (3H, m), 2.36-2.45 (1H,m), 3.54 (1H, dt, J=10.1, 3.5 Hz), 3.84 (3H, s, COOMe), 4.09 (1H, d,J=10.1 Hz). ¹³C NMR (CDCl₃) δ (ppm): 19.2, 25.3, 28.5, 38.7, 39.0, 43.6,44.1, 47.8, 49.9, 53.1, 176.7.

Method 6b

0.31 ml (4.57 mmol) of thionyl chloride was added dropwise with stirringto 4 ml of dry methanol keeping the internal temperature below −12° C.during the addition. After that, 0.91 g (4.17 mmol) of(1R,2R,3S,5R)-2-amino-6,6-dimethylbicyclo[3.1.1]heptan-3-carboxylic acidhydrochloride (6), prepared according to Example 5, was added to thesolution in one portion and the mixture was stirred for 30 min at 0° C.,3 h at room temperature, than for 30 min on boiling. The solution wasevaporated to dryness and the resulting yellow crude product wasrecrystallized from isopropyl ether/ethyl acetate mixture. The isolatedproduct 14 was: 0.78 g (80%).

EXAMPLE 7 Ethyl(1R,2R,3S,5R)-2-amino-6,6-dimethylbicyclo[3.1.1]heptan-3-carboxylatehydrochloride (compound 15) (Scheme 3)

Compound 15 was prepared by two methods:

Method 7a

1.16 g (7.0 mmol) of(1R,2R,5S,7R)-8,8-dimethyl-3-azatricyclo[5.1.1.0^(2,5)]nonan-4-one (10),prepared according to Example 1, was stirred in solution of 10%hydrochloric acid in dry ethanol (20 ml) at room temperature. After 1.5h stirring, the solution was evaporated to dryness and the crystallineproduct obtained was recrystallized from isopropyl ether/ethyl acetatemixture.

Isolated compound: 1.54 g (89%); mp: 138-139° C.; [α]_(D) ²⁰=+23.0(c=0.5, MeOH); IR=2918, 1729, 1373, 1189 cm⁻¹. Anal. Calcd. forC₁₂H₂₂ClNO₂ (247.76): C, 58.17; H, 8.95; N, 5.65. Found: C, 58.43; H,9.26; N, 5.51.¹H NMR (CDCl₃) δ (ppm): 0.97 (3H, s, Me-6), 1.36 (3H, s,Me-6), 1.35 (3H, t, CH₂-CH₃, J=7.3 Hz), 1.56 (1H, d, H-4, J=11.1 Hz),2.08-2.23 (3H, m), 2.35-2.46 (2H, m), 3.51 (1, dt, J=10.1, 3.0 Hz), 4.08(1H, d, J=9.6 Hz), 4.24-4.35 (2H, m, CH₂-CH₃). ¹³C NMR (CDCl₃) δ (ppm):13.7 (Me), 19.7 (Me), 24.4 (CH₂), 25.8 (CH), 29.4 (CH₂), 34.7 (CH) 39.4(C_(q)), 39.6 (Me), 44.2 (CH), 50.4 (CH), 63.0 (CH₂), 176.7 (C═O).

Method 7b

0.22 ml (3.05 mmol) of thionyl chloride was added dropwise with stirringto 4 ml of dry methanol keeping the internal temperature below −12° C.during the addition. After that, 0.61 g (2.78 mmol) of(1R,2R,3S,5R)-2-amino-6,6-dimethylbicyclo[3.1.1]heptan-3-carboxylic acidhydrochloride 6, prepared according to Example 5, was added to thesolution in one portion and the mixture was stirred for 30 min at 0° C.After standing for 3 h at room temperature the solution was boiled for30 min. The solution was evaporated to dryness and the resulted yellowcrude product was recrystallized from isopropyl ether/ethyl acetatemixture. The isolated product 15 was: 0.56 g (81%).

EXAMPLE 8 Ethyl(1S,2S,3R,5S)-2-amino-6,6-dimethylbicyclo[3.1.1]heptan-3-carboxylatehydrochloride (compound 34) (Scheme 7)

The synthesis of 1S,2S,3R,5S enantiomer 33 was accomplished by analogywith Example 7a, starting from 1S,2S,5R,7S enantiomer azetidinone 33;[α]^(D) ²⁰=−19.6 (c=0.5, MeOH); all the spectroscopic data and mp weresimilar to those for 1R,2R,3S,5R enantiomer 15.

EXAMPLE 9(1R,2R,3S,5R)-(2-tert-Butoxycarbonylamino)-6,6-dimethylbicyclo[3.1.1]heptan-3-carboxylicacid (compound 16) (Scheme 4)

0.66 g (3 mmol) of(1R,2R,3S,5R)-2-amino-6,6-dimethylbicyclo[3.1.1]heptan-3-carboxylic acidhydrochloride 6, prepared according to Example 5, was dissolved in 5 mlof distilled water at 0° C. and the pH of the solution was adjusted topH=8 with 3% sodium hydroxide solution in the presence of bromothymolblue indicator (the amfoter form of amino acid is precipitated off). 5ml of dioxane and 0.72 g (3.3 mmol) of Boc₂O was than added to themixture. The reaction mixture was stirred at room temperaturemaintaining the pH=8 with 3% sodium hydroxide solution. After 6 hstirring at room temperature, the solution was cooled to 0° C. andacidified with 5% hydrochloric acid solution to pH=5, then the solutionwas extracted with chloroform (3×50 ml). The combined organic layer wasdried (Na₂SO₄) and evaporated, and the white crystalline productobtained was recrystallized from n-hexane. The NMR measurement of theresulted product showed the present of approx. 12% isomer impurity(probably the trans compound) despite of the recrystallization processwas repeated several times.

Isolated compound: 0.52 g (61%); mp: 151-153° C.; [α]_(D) ²⁰=+46.0(c=0.5, MeOH); IR=3255, 2914, 1711, 1654, 1407, 1171 cm⁻¹. Anal. Calcd.for C₁₅H₂₅NO₄ (283.36): C, 63.58; H, 8.89; N, 4.94; Found: C, 63.79; H,8.41; N, 5.19. ¹H NMR (CDCl₃) δ (ppm): 0.88 (3H, s, Me-6), 1.24 (3H, s,Me-6), 1.47 (9H, s, CMe₃), 1.75 (1H, d, H-4, J =10.6 Hz), 1.84-1.99 (3H,m), 2.20-2.31 (2H, m), 3.21 (1H, dt, J=2.5, 10.1 Hz), 4.31 (1H, t,J=10.1 Hz), 7.33 (1H, d, J=10.1 Hz), 11.30 (1H, br s). ¹³C NMR (CDCl₃) δ(ppm): 20.6 (Me), 24.5 (CH₂), 26.5 (Me), 27.5 (CH2), 28.6 (CMe₃), 39.0(CH), 39.3 (C_(q)), 39.6 (CH), 46.7 (CH), 50.5 (CH), 81.4 (CMe₃), 155.8(NC=O), 179.8 (C═O ).

EXAMPLE 10(1S,2S,3R,5S)-(2-tert-Butoxycarbonylamino)-6,6-dimethylbicyclo[3.1.1]heptan-3-carboxylicacid (compound 37) (Scheme 7)

The synthesis of 1S,2S,3R,5S enantiomer 37 was accomplished by analogywith Example 9, starting from 1S,2S,3R,5S enantiomer amino acidhydrochloride 7; [α]_(D) ²⁰=−44.1 (c=0.5, MeOH); all the spectroscopicdata and mp were similar to those for (1R,2R,3S,5R) enantiomer 16.

The synthesis of1S,2S,3R,5S-2-amino-6,6-dimethylbicyclo[3.1.1]heptan-3-carboxylic acidhydrochloride 7 was accomplished by analogy with Example 5, startingfrom 1S,2S,5R,7S enantiomer azetidinone 33; [α]_(D) ²⁰=−21.6 (c=0.5,MeOH); all the spectroscopic data and mp were similar to those for1R,2R,3S,5R enantiomer 6.

EXAMPLE 11 Ethyl(2S,1′R,2′R,3′S,5′R)-2-[(2′-tert-butoxycarbonylamino)-6′,6′-dimethylbicyclo[3.1.1]heptan-3′carbonyl)]amino-3-phenylpropionate(compound 17) (Scheme 4)

0.05 g of triethylamine and 0.065 g of isobutyl chloroformate were addedto the solution of 0.14 g (0.49 mmol) of (1R,2R,3S,5R) Boc protectedamino acid 16, prepared according to Example 9, in 5 ml of dry THE at−10° C. with vigorous stirring. After 10 min stirring and cooling, 2 mldry THF solution of 0.095 g (0.49 mmol) of phenylalanine ethyl ester wasadded dropwise to the mixture at −10° C. The mixture was stirred at roomtemperature for father 5 h, than evaporated to dryness. The resultingoily crude product was dissolved in chloroform (30 ml) and the organicsolution was washed first with ice-cold 5% solution of sodium hydrogencarbonate (20 ml), than with ice-cold 5% hydrochloric acid solution (20ml). The organic layer was dried (Na₂SO₄) and evaporated. The obtainedoily product was purified by flash chromatography on a silica gel column(n-hexane:ethyl acetate=6:1, Rf=0.35).

Isolated compound: 0.15 g (67%), viscous oil; [α]_(D) ²⁰=+22.5 (c=0.5,MeOH); IR=3411, 2978, 1711, 1497, 1366, 1161, 701 cm⁻¹. Anal. Calcd. forC₂₆H₃₈N₂O₅ (458.59): C, 68.10; H, 8.35; N, 6.11. Found: C, 67.76; H,8.53; N, 6.38. ¹H NMR (CDCl₃) δ (ppm): 0.88 (3H, s, Me), 1.22 (6H, soverlapped with t), 1.36 (9H, s, CMe₃), 1.62-2.20 (7H, m), 3.01 (1H, dt,J=3.5, 10.1 Hz), 3.15 (2H, ddd, J=3.5, 10.1, 42.3 Hz), 4.14 (2H, q,J=7.1, 14.1 Hz), 4.35 (1H, t, J=10.0 Hz), 4.74 (1H, q, J=6.1, 12.1 Hz),5.28 (1H, d, J=10.1 Hz), 6.19 (1H, d, J=6.5 Hz), 7.9 (2H, d, J=7.1 Hz),7.20-7.30 (3H, m). ¹³C NMR (CDCl₃) δ (ppm): 14.7 (Me), 20.9 (Me), 25.4(CH₂), 26.8 (Me), 29.0 (CMe₃), 29.8 (CH₂), 38.6 (CH₂), 39.8 (C_(q)),40.0 (Me), 40.5 (CH), 46.9 (CH), 49.5 (CH), 54.5 (CH), 62.1 (CH₂), 79.6(CMe₃), 127.7 (CH_(ar)), 129.1 (CH_(ar)), 130.1 (CH_(ar)), 136.7(C_(q)), 156.1 (C═O, Boc), 171.6 (C═O ), 175.4 (C═O).

EXAMPLE 12 Ethyl(2S,1′S,2′S,3′R,5′S)-2-[(2′-tert-butoxycarbonylamino)-6′,6′-dimethylbicyclo[3.1.1]heptan-3′-carbonyl)]amino-3-phenylpropionate(compound 38) (Scheme 7)

The synthesis of 1S,2S,3R,5S enantiomer 38 was accomplished by analogywith Example 11, starting from 0.14 g (0.49 mmol) 1S,2S,3R,5S enantiomer37 prepared according to Example 10.

Isolated compound: 0.12 g (54%); oil; [α]_(D) ²⁰=−10.0 (c=0.25, MeOH);IR=3306, 2923, 2852, 1744, 1681, 1500, 1330, 1160, 1042 cm⁻¹. Anal.Calcd. for C₂₆H₃₈N₂O₅ (458.59): C, 68.10; H, 8.35; N, 6.11. Found: C,68.45; H, 8.01; N, 6.43. ¹H NMR (CDCl₃) δ (ppm): 0.90 (3H, s, Me-6),1.15 (3H, t, CH₂-CH₃, J=7.1 Hz), 1.23 (3H, s, Me-6), 1.38 (9H, s), 1.74(1H, d, J=10.1 Hz), 1.87-2.21 (5H, m), 2.93 (1H, dd, J=14.1, 7.1 Hz),3.03 (1H, dt, J=10.1, 4.0 Hz), 3.21 (1H, dd, J=14.1, 5.0 Hz), 4.09 (2H,dd, J=14.1, 7.1 Hz), 7.10-7.31 (5H, m). ¹³C NMR (CDCl₃) δ (ppm): 14.7,20.9, 25.5, 26.9, 29.2, 29.8, 30.4, 39.3, 40.0, 40.4, 47.2, 49.6, 54.2,62.0, 79.9, 127.8, 129.3, 130.0, 136.4, 156.1, 172.0, 175.3.

EXAMPLE 13(1R,2R,3S,5R)-2-(9H-Fluoren-9-yl-methoxycarbonylamino)-6,6-dimethylbicyclo[3.1.1]heptan-3-carboxylicacid (compound 18) (Scheme 4)

0.34 g (1.56 mmol) of(1R,2R,3S,5R)-2-amino-6,6-dimethylbicyclo[3.1.1]heptan-3-carboxylic acidhydrochloride (6), prepared according to Example 5, was dissolved in 6ml of distilled water at 0° C. 0.50 g (6 mmol) of sodium hydrogencarbonate, 5 ml of acetonitrile and 0.51 g (1.5 mmol) of Fmoc-Osu wasgiven to the solution at 0° C. After 12 h stirring at room temperature,the pH of the solution was adjusted to pH=2 with 10% hydrochloric acidsolution and after 1 h stirring, the reaction mixture was extracted withethyl acetate (3×20 ml). The combined organic layer was dried (Na₂SO₄),evaporated, and the crude product obtained was purified by flashchromatography on a silica gel column (n-hexane:ethyl acetate=9:1,Rf=0.35) resulting in white crystalline product.

Isolated compound: 0.40 g (63%); mp: 160-162° C.; [α]_(D) ²⁰=+2.0(c=0.25, MeOH); IR=3260, 2908, 1711, 1652, 1414, 1332, 1201, 741 cm⁻¹.Anal. Calcd. for C₂₅H₂₇NO₄ (405.49): C, 74.05; H, 6.71; N, 3.45. Found:C, 74.19; H, 6.45; N, 3.53. ¹H NMR (CDCl₃) δ (ppm): 0.84 (3H, s), 1.23(3H, s), 1.71 (1H, d, J=10.4 Hz), 1.81-2.25 (5H, m), 3.08 (1H, t, J=9.1Hz), 4.00-4.47 (4H, m), 7.24-7.75 (8H, m). ¹³C NMR (CDCl₃) δ (ppm):20.8, 24.8, 26.8, 28.8, 39.1, 39.6, 39.9, 46.9, 47.9, 50.8, 68.2, 120.6,125.6, 125.9, 127.7, 128.3, 142.0, 142.1, 144.5, 144.8 158.7, 180.2.

EXAMPLE 14(1S,2S,3R,5S)-2-(9H-Fluoren-9-yl-methoxycarbonylamino)-6,6-dimethylbicyclo[3.1.1]heptan-3-carboxylicacid (compound 36) (Scheme 7)

The synthesis of 1S,2S,3R,5S enantiomer 36 was accomplished by analogywith Example 13, starting from 1S,2S,3R,5S enantiomer amino acidhydrochloride (7) (prepared according to Example 10); [α]_(D) ²⁰=−2.0(c=0.25, MeOH); all the spectroscopic data and mp were similar to thosefor 1R,2R,3S,5R enantiomer 18.

EXAMPLE 15(1S,2S,3R,5S)-2-Dimethylamino-6,6-dimethylbicyclo[3.1.1]heptan-3-carboxylicacid hydrochloride (compound 19) (Scheme 4)

0.50 g (2.27 mmol) of amino acid hydrochloride 6 prepared according toExample 5, was dissolved in 15 ml of distilled water, followed byaddition of 0.43 g (5.0 mmol) of 35% formaldehyde solution and 0.20 g of10% Pd/C catalyst. The mixture was stirred at room temperature and 10bar under H₂ atmosphere for 12 h. The mixture was than filtered andevaporated to dryness. The resulting crystalline product was rubbed withacetone, than filtered off.

Isolated compound: 0.50 g (89%); mp: 144-145° C.; [α]_(D) ²⁰=+10.7(c=0.505, MeOH); IR=2952, 1709, 1458, 1186 cm⁻¹. Anal. Calcd. forC₁₂H₂₂ClNO₂ (247.76): C, 58.17; H, 8.95; N, 5.65. Found: C, 58.23; H,8.69; N, 5.37. ¹H NMR (CDCl₃) δ (ppm): 0.89 (3H, s, Me-6), 1.35 (3H, s,Me-6), 1.62 (1H, d, H-4, J=11.1 Hz), 2.03-2.10 (1H, m), 2.21-2.56 (4H,m), 2.88 (3H, s), 2.92 (3H, s), 3.51 (1H, t, J=9.3 Hz), 3.76 (1H, d,J=8.6 Hz). ¹³C NMR (CDCl₃) δ (ppm): 19.4 (Me), 23.4 (CH₂), 25.9 (Me),29.5 (CH₂), 34.3 (CH), 39.3 (CH), 40.3 (CH), 40.4 (C_(q)), 42.3 (Me),44.1 (Me), 67.2 (CH), 179.9 (C═O).

EXAMPLE 16 Ethyl(1R,2R,3S,5R)-2-benzylamino-6,6-dimethylbicyclo[3.1.1]heptan-3-carboxylatehydrochloride (compound 20) (Scheme 5)

1.05 g (5 mmol) of ethyl(1R,2R,3S,5R)-2-amino-6,6-dimethylbicyclo[3.1.1]heptan-3-carboxylate(15) base, prepared according to Example 7a, and 0.53 g (5 mmol) ofbenzaldehyde was dissolved in 30 ml of dry ethanol. The solution wasstirred at room temperature for 2 h, than evaporated to dryness. Theresulting viscous oil was dissolved in 30 ml of dry ethanol, followed byaddition of 0.57 g (0.015 mmol) of sodium borohydride in small portionsat 0° C. After 6 h stirring at room temperature, the solution wasevaporated to dryness, the residual was dissolved in 50 ml of ice-coldwater and extracted with chloroform (3×50 ml). The combined organiclayer was dried (Na₂SO₄) and evaporated, and the hydrochloride salt 20,prepared from the amino ester base with 10% solution of hydrochloricacid in dry ethanol, was recrystallized from isopropyl ether.

Isolated compound: 1.18 g (70%); mp: 165-168° C.; [α]_(D) ²⁰=+16.4(c=0.525, MeOH); IR=2919, 1733, 1445, 1183, 705 cm⁻¹. Anal. Calcd. forC₁₉H₂₈ClNO₂ (337.88): C, 67.54; H, 8.35; N, 4.15. Found: C, 67.79; H,8.21; N, 4.47. ¹H NMR (CDCl₃) δ (ppm): 0.91 (3H, s, Me-6), 1.34 (3H, t,CH₂-CH₃, J=7.1 Hz), 1.37 (3H, s, Me-6), 1.63 (1H, d, H-4, J=10.1 Hz),2.07-2.11 (1H, m), 2.16-2.23 (1H, m), 2.36-2.51 (3H, m), 3.46 (1H, dt,J=10.1, 2.5 Hz), 3.97 (1H, d, J=9.1 Hz), 4.19-4.37 (4H, m, CH₂-CH₃ andCH₂Ph), 7.52-7.58 (5H, m, Ph). ¹³C NMR (CDCl₃) δ (ppm): 13.6 (Me), 19.4(Me), 24.0 (CH₂), 25.8 (CH), 29.5 (CH₂), 33.9 (CH), 39.7 (Me), 40.1(C_(q)), 42.4 (CH), 50.4 (CH₂), 57.4 (CH), 63.2 (CH₂), 129.8 (CH), 130.2(CH), 130.3 (CH), 131.0 (C_(q)), 177.6 (C═O).

EXAMPLE 17(1S,2S,3R,5S)-2-Benzylamino-6,6-dimethylbicyclo[3.1.1]heptan-3-carboxylicacid hydrochloride (compound 21) (Scheme 5)

0.80 g (2.37 mmol) of compound 20, prepared according to Example 16, wasdissolved in 40 ml of 10% solution of hydrochloric acid. The mixture wasstirred at room temperature while the hydrolysis was accomplished (theprogress of the reaction was monitored by means of ¹H NMR). When thehydrolysis was accomplished, the solution was evaporated to dryness andthe resulting crystalline product was rubbed with acetone, than filteredoff.

Isolated compound: 0.46 g (63%); mp: 171-172° C.; [α]_(D) ²⁰=+12.9(c=0.52, MeOH); IR=2918, 1697, 1456, 1200, 697 cm⁻¹. Anal. Calcd. forC₁₇H₂₄ClNO₂ (309.83): C, 65.90; H, 7.81; N, 4.52. Found: C, 65.83; H,8.12; N, 4.77. ¹H NMR (CDCl₃) δ (ppm): 0.89 (3H, s, Me-6), 1.35 (3H, s,Me-6), 1.64 (1H, d, H-4, J=11.1 Hz), 2.06-2.12 (1H, m), 2.18-2.25 (1H,m), 2.32-2.53 (3H, m), 3.35-3.42 (1H, m), 3.93 (1H, d, J=9.1 Hz), 4.27(2H, dd, J=39.3, 13.6 Hz, CH₂Ph), 7.48-7.57 (5H, m, Ph). ¹³C NMR (CDCl₃)δ (ppm): 19.4 (Me), 23.9 (CH₂), 25.7 (Me), 29.7 (CH₂), 33.6 (CH), 39.7(CH), 40.1 (C_(q)), 42.6 (CH), 50.3 (CH₂), 57.2 (CH), 129.7 (CH), 130.2(CH), 130.3 (CH), 130.9 (C_(q)), 179.7 (C═O).

EXAMPLE 18(1R,2R,3S,5R)-(2-Amino-6,6-dimethylbicyclo[3.1.1]hept-3-yl)-methanolhydrochloride (compound 22) (Scheme 5)

To a slurry of LiAlH₄ 0.93 g (24.5 mmol) in 150 ml of dry THF, 2.00 g(9.5 mmol) of (1R,2R,3S,5R) amino ester liberated from compound 15(prepared according to Example 7a) was added dropwise at 0° C. Afterstirring at room temperature for 1.5 h (the reduction was monitored bymeans of TLC), the mixture was decomposed with the mixture of 10 ml ofTHF and 2.0 ml of water under ice cooling. The inorganic material wasfiltered off and washed with THF. After drying (Na₂SO₄) and evaporation,pale-yellow oil was obtained. The amino alcohol obtained was purified asthe hydrochloride with recrystallizing from diethyl ether/ethanolmixture.

Isolated compound: 1.45 g (70%): mp: 179-183° C.; [α]_(D) ²⁰=−16.4(c=0.5, MeOH); IR=3123, 2917, 1529, 1457, 1051 cm⁻¹. Anal. Calcd. forC₁₀H₂₀ClNO (205.72): C, 58.38; H, 9.80; N, 6.81. Found: C, 58.61; H,10.11; N, 6.49. ¹H NMR (CDCl₃) δ (ppm): 0.95 (3H, s, Me-6), 1.15 (1H, d,H-4, J=11.1 Hz), 1.28 (3H, s, Me-6), 1.44 (1H, dt, J=4.0, 14.1 Hz, H-7),1.97-2.03 (1H, m, H-5), 2.09-2.18 (2H, m, H-1, H-7), 2.27-2.34 (1H, m,H-4), 2.59-270 (1H, m, H-3), 3.73 (2H, ddd, J=5.0, 11.58, 40.2 Hz,CH₂—OH), 3.98 (1H, d, J=9.6 Hz, H-2). ¹³C NMR (CDCl₃) δ (ppm): 19.9(Me), 25.2 (CH₂), 25.8 (Me), 29.0 (CH₂), 30.3 (CH), 38.6 (C_(q)), 40.0(CH), 45.0 (CH), 52.9 (CH), 65.0 (CH₂).

EXAMPLE 19(1S,2S,3R,5S)-(2-Amino-6,6-dimethylbicyclo[3.1.1]hept-3-yl)-methanolhydrochloride (compound 35) (Scheme 7)

The synthesis of 1S,2S,3R,5S enantiomer 35 was accomplished by analogywith Example 18, starting from 1S,2S,3R,5S enantiomer amino ester 34which was prepared according to Example 8; [α]_(D) ²⁰=+13.4 (c=0.5,MeOH); all the spectroscopic data and mp were similar to those for1R,2R,3S,5R enantiomer 22.

EXAMPLE 20(1R,2R,3S,5R)-(6,6-Dimethyl-2-methylaminobicyclo[3.1.1]heptan-3-yl)-methanolhydrochloride (compound 23) (Scheme 5)

To a slurry of LiAlH₄ 2.82 g (74.32 mmol) in 150 ml of dry THF, 10 mlTHF solution of 4.78 g (16.9 mmol) of N-Boc amino acid 16 (preparedaccording to Example 9) was added dropwise at room temperature. Afterstirring at room temperature for 6 h (the reduction was monitored bymeans of TLC), the mixture was decomposed with the mixture of 30 ml ofTHF and 6.0 ml of water under ice cooling. After one hour of standingthe inorganic material was filtered off and washed with THF. Afterdrying (Na₂SO₄) and evaporation, a pale-yellow oil was obtained. Theamino alcohol was purified as the hydrochloride with recrystallizingfrom diethyl ether/ethanol mixture.

Isolated compound: 1.44 g (39%): mp: 192-193° C.; [α]_(D) ²⁰=−15.7(c=0.5, MeOH);

IR=3308, 3123, 2916, 2475, 1595, 1458, 1049 cm⁻¹. Anal. Calcd. forC₁₁H₂₂ClNO (219.14): C, 60.12; H, 10.09; N, 7.20. Found: C, 60.33; H,10.27; N, 6.95. ¹H NMR (D₂O) δ (ppm): 0.99 (3H, s), 1.18 (1H, d, J=10.6Hz), 1.35 (3H, s), 1.48-1.55 (1H, m), 2.03-2.10 (1H, m), 2.15-2.24 (1H,m), 2.33-2.47 (2H, m), 2.74 (3H, s), 2.72-280 (1H, m), 3.73-3.91 (3H,m). ¹³C NMR (CDCl₃) δ (ppm): 20.0 (Me), 25.1 (CH₂), 26.1 (Me), 29.2(CH₂), 31.1 (CH), 32.1 (CH), 38.4 (C), 39.9 (CH), 41.1 (CH), 62.0 (Me),65.1 (CH₂).

EXAMPLE 21(1R,2R,3S,5R)-(2-Benzylamino-6,6-dimethylbicyclo[3.1.1]heptan-3-yl)-methanolhydrochloride (compound 24) (Scheme 5)

To a slurry of LiAlH₄ 0.78 g (20.4 mmol) in 50 ml of dry THF, 25 ml THFsolution of 3.06 g (10.2 mmol) of 1R,2R,3S,5R amino ester 20 (preparedaccording to Example 16) was added dropwise at room temperature. Afterstirring at room temperature for 4 h (the reduction was monitored bymeans of TLC), the mixture was decomposed with the mixture of 20 ml ofTHF and 2.0 ml of water under ice cooling. The inorganic material wasfiltered off and washed with THE After drying (Na₂SO₄) and evaporation,a pale-yellow oil was obtained. The prepared amino alcohol was purifiedas the hydrochloride with recrystallizing from diethyl ether/ethanolmixture.

Isolated compound: 1.82 g (61%): mp: 252-253° C.; [α]_(D) ²⁰=−8.5(c=0.5, MeOH); IR=3177, 2927, 2741, 1597, 1457, 1048 cm⁻¹. Anal. Calcd.for C₁₇H₂₆ClNO (295.85): C, 69.02; H, 8.86; N, 4.73. Found: 68.85; H,8.67; N, 4.97. ¹H NMR (D₂O) δ (ppm) 0.94 (3H, s, Me-6), 1.23 (1H, d,H-4, J=11.1 Hz), 1.36 (3H, s, Me-6), 1.44 (1H, dt, J=4.0, 14.1 Hz, H-7),2.02-2.23 (2H, m), 2.37-2.55 (3H, m), 2.64-2.75 (1H, m), 3.74-3.94 (3H,m), 4.23 (1H, d, J=13.1), 4.44 (1H, d, J=13.1 Hz). ¹³C NMR (CDCl₃) δ(ppm): 19.8 (Me), 25.3 (CH₂), 25.9 (Me), 28.8 (CH₂), 30.7 (CH), 38.5(C_(q)), 39.7 (CH), 41.9 (CH), 49.9 (CH₂), 59.8 (CH), 65.6 (CH₂), 129.8(4×CH), 130.1 (CH), 131.5 (C_(q)).

EXAMPLE 22(1R,2R,3S,5R)-(2-Dimethylamino-6,6-dimethylbicyclo[3.1.1]hept-3il)-methanolhydrochloride (compound 25) (Scheme 5)

3.50 g (20.7 mmol) of the base of amino alcohol hydrochloride 22,prepared according to Example 18, was dissolved in a mixture of 40 ml offormic acid and 40 ml of 35% formaldehyde solution. The mixture wasrefluxed for 1 h and, after cooling down, the solution was made alkalinewith 20% aqueous KOH and extracted with chloroform (3×100 ml). Thecombined organic phase was dried (Na₂SO₄) and evaporated to give 2.50 gof an almost colourless oil which was used in the next step withoutfurther purification. To a slurry of 1.94 g (51.2 mmol) of LiAlH₄ in 100ml of dry THF, the oily product obtained above in 10 ml of dry THF wasadded dropwise at room temperature After stirring and refluxing for 3 h(the reduction was monitored by means of TLC), the mixture wasdecomposed with the mixture of 20 ml of THF and 4.0 ml of water underice cooling. After stirring for 1 h, the inorganic material was filteredoff and washed with THF (2×100 ml). After drying (Na₂SO₄) andevaporation, a pale-yellow oil was obtained. The amino alcohol 25 waspurified as the hydrochloride recrystallizing from diethyl ether/ethanolmixture.

Isolated compound: 2.45 g (51%): mp: 176-177° C.; [α]_(D) ²⁰=+0.9(c=0.5, MeOH); IR=3332, 2924, 2711, 1484, 1042 cm⁻¹. Anal. Calcd. forC₁₂H₂₄ClNO (233.78): C, 61.65; H, 10.35; N, 5.99. Found: C, 61.84; H,10.12; N, 6.19. ¹H NMR (CDCl₃) δ (ppm): 0.83 (3H, s, Me-6), 1.27 (3H, s,Me-6), 1.46 (1H, d, H-4, J=10.6 Hz), 1.57-1.64 (2H, m), 1.83-1.99 (2H,m), 2.19-2.27 (1H, m), 2.34 (1H, t, J=5.0 Hz), 2.71 (3H, br s), 2.82(3H, br s), 3.67-3.75 (2H, m), 3.79-3.86 (1H, m), 5.83 (1H, br s), 9.68(1H, br s). ¹³C NMR (CDCl₃) δ (ppm): 19.9 (2×Me), 25.0 (CH₂), 26.0 (CH),27.0 (CH₂), 31.4 (2×Me), 38.7 (CH), 39.1 (CH), 39.8 (C_(q)), 64.1 (CH₂),66.9 (CH).

EXAMPLE 23 Ethyl(1R,2R,3R,5R)-2-amino-6,6-dimethylbicyclo[3.1.1]heptan-3-carboxylatehydrochloride (compound 26) (Scheme 6)

To a solution of 0.23 g (10 mmol) of sodium in 30 ml of dry ethanol,1.05 g (5 mmol) base of ethyl(1R,2R,3S,5R)-2-amino-6,6-dimethylbicyclo[3.1.1]heptan-3-carboxylate(15), prepared according to Example 7, was added in one portion. Thesolution was stirred at room temperature until isomerisation wasaccomplished (approx. 4 h, the isomerisation process was monitored bymeans of TLC and GC). The solution was evaporated to approx. 5 ml,diluted with ice-cold water (50 ml) and extracted with ethyl acetate(3×50 ml). The combined organic layer was dried (Na₂SO₄) and evaporated,and the hydrochloride salt 26, prepared from the resulted amino esterbase with 15% solution of hydrochloric acid in dry ethanol, wasrecrystallized from isopropyl ether.

Isolated compound: 0.85 g (69%); mp: 147-148° C.; [α]_(D) ²⁰=−32.4(c=0.5, MeOH); IR=2926, 1734, 1509, 1292, 1193 cm⁻¹. Anal. Calcd. forC₁₂H₂₂ClNO₂ (247.76): C, 58.17; H, 8.95; N, 5.65. Found: C, 58.35; H,8.78; N, 5.79. ¹H NMR (CDCl₃) δ (ppm): 0.88 (3H, s, Me-6), 1.34 (3H, s,Me-6), 1.36 (3H, t, CH₂-CH₃, J=7.1 Hz), 1.56 (1H, d, H-4, J=11.1 Hz),2.05 (1H, dd, J=13.6, 9.1 Hz), 2.10-2.23 (2H, m), 2.35-2.46 (2H, m),3.05 (1H, dd, J=9.1, 18.1 Hz), 4.10 (1H, d, J=89.6 Hz), 4.33 (2H, dd,J=7.1, 14.1 Hz, CH₂-CH₃). ¹³C NMR (CDCl₃) δ (ppm): 13.8 (Me), 19.1 (Me),23.0 (CH₂), 25.8 (CH), 27.5 (CH₂), 38.4 (CH), 39.3 (Me), 39.7 (C_(q)),43.4 (CH), 52.3 (CH), 63.0 (CH₂), 175.9 (C═O).

EXAMPLE 24 Ethyl(1S,2S,3S,5S)-2-amino-6,6-dimethylbicyclo[3.1.1]heptan-3-carboxylatehydrochloride (compound 39) (Scheme 8)

The synthesis of 1S,2S,3S,5S enantiomer 39 was accomplished by analogywith Example 23, starting from 1S,2S,3R,5S enantiomer amino ester 34which was prepared according to Example 8; [α]_(D) ²⁰=+31.0 (c=0.5,MeOH); all the spectroscopic data and mp were similar to those for1R,2R,3R,5R enantiomer 26.

EXAMPLE 25 Ethyl(1R,2R,3R,5R)-2-benzylamino-6,6-dimethylbicyclo[3.1.1]heptan-3-carboxylatehydrochloride (compound 27) (Scheme 6)

1.05 g (5 mmol) of ethyl(1R,2R,3R,5R)-2-amino-6,6-dimethylbicyclo[3.1.1]heptan-3-carboxylate(26) base prepared according to Example 23, and 0.53 g (5 mmol) ofbenzaldehyde was dissolved in 30 ml of dry ethanol. The solution wasstirred at room temperature for 2 h, than evaporated to dryness. Theresulting viscous oil was dissolved in 30 ml of dry ethanol, followed byaddition of 0.57 g (0.015 mmol) of sodium borohydride in small portionsat 0° C. After 6 h stirring at room temperature, the solution wasevaporated to dryness, the resulting mass was dissolved in 50 ml ofice-cold water and extracted with chloroform (3×50 ml). The combinedorganic layer was dried (Na₂SO₄) and evaporated, and the hydrochloride27, prepared from the amino ester base with 10% solution of hydrochloricacid in dry ethanol, was recrystallized from isopropyl ether.

Isolated compound: 1.23 g (73%); mp: 134-136° C.; [α]_(D) ²⁰=−0.25,MeOH); ER=2948, 2778, 1731, 1577, 1182, 747 cm⁻¹. Anal. Calcd. forC₁₉H₂₈ClNO₂ (337.88): C, 67.54; H, 8.35; N, 4.15. Found: C, 67.79; H,8.21; N, 4.47. ¹H NMR (CDCl₃) δ (ppm): 0.71 (3H, s), 1.22 (3H, t, J=7.1Hz), 1.30 (3H, s), 1.53 (1H, d, J=10.6 Hz), 1.89 (1H, dd, J=13.6, 8.8Hz), 2.02-2.09 (1H, m), 2.31-2.42 (3H, m), 3.08 (1H, dd, J=18.8, 8.8Hz), 3.89 (1H, d, J=8.4 Hz), 4.10-4.22 (1H, m), 4.27 (2H, dd, J=42.7,13.3 Hz), 7.45-7.56 (5H, m, Ph). ¹³C NMR (CDCl₃) δ (ppm): 13.8, 19.0,23.4, 25.9, 28.0, 37.8, 39.4, 39.7, 40.5, 49.1, 57.6, 64.8, 129.8,130.3, 130.6, 131.0, 175.5.

EXAMPLE 26(1R,2R,3R,5R)-2-Amino-6,6-dimethylbicyclo[3.1.1]heptan-3-carboxylic acid(compound 29) (Scheme 6)

Method 26a

0.23 g (1.09 mmol) of base liberated from ethyl(1R,2R,3R,5R)-2-amino-6,6-dimethylbicyclo[3.1.1]heptan-3-carboxylatehydrochloride 26 (prepared according to Example 23) was dissolved in themixture of 10 ml of dioxane and 10 ml of distilled water and thesolution was heated at 80° C. with monitoring by means of TLC. When thereaction was accomplished (indicated by elimination of the startingester), the mixture was evaporated to dryness and the resulting whitecrystalline product was rubbed with acetone, filtered off andrecrystallized from acetone/water mixture.

Isolated compound: 0.13 g (65%); mp: 250-252° C.; [α]_(D) ²⁰=−42.7(c=0.5, MeOH); IR=2924, 1624, 1552, 1404 cm⁻¹. Anal. Calcd. forC₁₀H₁₇NO₂ (183.25): C, 65.54; H, 9.35; N, 7.64. Found: 65.21; H, 9.87;N, 7.19. ¹H NMR (CDCl₃) δ (ppm): 0.82 (3H, s, Me-6), 1.26 (3H, s, Me-6),1.49 (1H, d, J=10.8 Hz), 1.83-1.93 (1H, m), 1.98-2.11 (2H, m), 2.20-2.30(2H, m), 2.61-2.70 (1H, m), 3.90 (1H, d, J=8.6 Hz). ¹³C NMR (CDCl₃) δ(ppm): 19.1 (Me), 22.8 (CH₂), 25.8 (Me), 28.1 (CH₂), 39.7 (CH), 39.9(C_(q)), 40.6 (CH), 43.4 (CH), 53.6 (CH), 181.5 (C═O).

Method 26b

0.56 g (85%) of(1R,2R,3R,5R)-2-amino-6,6-dimethylbicyclo[3.1.1]heptan-3-carboxylic acidhydrochloride 8 was prepared according to Example 17, starting from 0.74g (3.0 mmol) of ethyl(1R,2R,3R,5R)-2-amino-6,6-dimethylbicyclo[3.1.1]heptan-3-carboxylatehydrochloride (26) (prepared according to Example 23).

Compound 8: mp: 241-242° C.; [α]_(D) ²⁰=−32.6 (c=0.5, MeOH); IR=2913,1716, 1511, 1247 cm⁻¹. Anal. Calcd. for C₁₀H₁₈ClNO₂ (219.71): C, 54.67;H, 8.26; N, 6.38. Found: C, 54.79; H, 8.39; N, 6.21. ¹H NMR (CDCl₃) δ(ppm): 0.88 (3H, s, Me-6), 1.34 (3H, s, Me-6), 1.56 (1H, d, J=11.1 Hz),2.05 (1H, dd, J=13.8, 8.8 Hz), 2.13 (1H, dd, J=9.8, 4.8 Hz), 2.20 (1H,t, J=5.5 Hz), 2.35-2.45 (2H, m), 2.99-3.07 (1H, m), 4.08 (1H, d, J=8.6Hz). ¹³C NMR (CDCl₃) δ (ppm): 19.1 (Me), 23.0 (CH₂), 25.8 (Me), 27.4(CH₂), 38.2 (CH), 39.4 (CH), 39.8 (C_(q)), 43.4 (CH), 52.4 (CH), 177.7(C═O).

0.56 g (2.58 mmol) of(1R,2R,3R,5R)-2-amino-6,6-dimethylbicyclo[3.1.1]heptan-3-carboxylic acidhydrochloride (8) was dissolved in 8 ml of distilled water and then pHof the solution was adjusted to pH=7.4 with 10% solution of NaHCO₃ withice cooling in the presence of methylthymolblue indicator. After 1 hstirring at 0° C. the precipitated crystalline product was filtered andwashed with a small amount of ice-cold distilled water. Isolatedcompound: 0.30 g (65%);

EXAMPLE 27(1R,2R,3R,5R)-(2-tert-Butoxycarbonylamino)-6,6-dimethylbicyclo[3.1.1]heptan-3-carboxylicacid (compound 31) (Scheme 6)

The synthesis of compound 31 was accomplished by analogy with Example 9,starting from 0.66 g (3 mmol) of(1R,2R,3R,5R)-2-amino-6,6-dimethylbicyclo[3.1.1]-heptan-3-carboxylicacid hydrochloride 8, which was prepared according to Example 26b.

Isolated compound: 0.55 g (65%); mp: 85-87° C.; [α]_(D) ²⁰=−43.3 (c=0.5,MeOH); IR=3336, 2923, 1711, 1659, 1366, 1176 cm⁻¹. Anal. Calcd. forC₁₅H₂₅NO₄ (283.36): C, 63.58; H, 8.89; N, 4.94. Found: C, 63.84; H,8.99; N, 5.26. ¹H NMR (CDCl₃) δ (ppm): 0.94 (3H, s), 1.23 (3H, s), 1.35(1H, d, J =10.1 Hz), 1.43 (9H, s), 1.94-2.21 (5H, m), 2.53-2.64 (1H, m),4.26-4.36 (1H, m), 4.73 (1H, br s). ¹³C NMR (CDCl₃) δ (ppm): 20.1, 24.1,27.2, 28.4, 29.0, 40.2, 40.4, 42.7, 46.7, 52.1, 81.0, 155.7, 179.8.

EXAMPLE 28(1S,2S,3S,5S)-(2-tert-Butoxycarbonylamino)-6,6-dimethylbicyclo[3.1.1]heptan-3-carboxylicacid (compound 42) (Scheme 8)

The synthesis of1S,2S,3S,5S-2-amino-6,6-dimethylbicyclo[3.1.1]heptan-3-carboxylic acidenantiomer 9 was accomplished by analogy with Example 19, starting from1S,2S,3S,5S enantiomer amino ester 39, prepared according to Example 24;[α]_(D) ²⁰=+30.1 (c=0.5, MeOH); all the spectroscopic data and mp weresimilar to those for 1R,2R,3R,5R enantiomer 8.

The synthesis of 1S,2S,3S,5S enantiomer 42 was accomplished by analogywith Example 9, starting from 0.66 g (3 mmol) of(1S,2S,3S,5S)-2-amino-6,6-dimethylbicyclo[3.1.1]heptan-3-carboxylic acidhydrochloride 9 prepared above, [α]_(D) ²⁰+41.1 (c =0.5, MeOH); all thespectroscopic data and mp were similar to those for 1R,2R,3R,5Renantiomer 31.

EXAMPLE 29(1R,2R,3R,5R)-(2-Amino-6,6-dimethylbicyclo[3.1.1]hept-3-yl)-methanolhydrochloride(compound 28) (Scheme 6)

To a slurry of LiAlH₄ 0.93 g (24.5 mmol) in 150 ml of dry THF, 2.00 g(9.5 mmol) of (1R,2R,3R,5R) amino ester liberated from compound 26(prepared according to Example 23) was added dropwise at 0° C. Afterstirring at room temperature for 1.5 h (the reduction was monitored bymeans of TLC), the mixture was decomposed with the mixture of 10 ml ofTHF and 2.0 ml of water under ice cooling. After 1 h stirring at roomtemperature, the inorganic material was filtered off and washed withTHF. After drying (Na₂SO₄) and evaporation, the amino alcohol obtainedas a pale-yellow oil was purified as the hydrochloride withrecrystallizing from diethyl ether/ethanol mixture.

Isolated compound: 1.62 g (78%); mp: 199-202° C.; [α]_(D) ²⁰−7.9(c=0.52, MeOH); IR=3298, 2905, 1512, 1040 cm⁻¹. Anal. Calcd. forC₁₀H₂₀ClNO (205.72): C, 58.38; H, 9.80; N, 6.81. Found: C, 58.49; H,9.71; N, 6.93. ¹H NMR (CDCl₃) δ (ppm): 0.86 (3H, s), 1.32 (3H, s),1.50-1.59 (1H, m), 1.62 (1H, d, J=10.6 Hz), 2.03-2.20 (4H, m), 2.28-2.36(1H, m), 3.63 (1H, d, J=8.1 Hz), 3.73 (2H, ddd, J=2.5, 5.5, 11.1 Hz).¹³C NMR (CDCl₃) δ (ppm): 19.1 (Me), 23.1 (CH₂), 26.2 (Me), 26.3 (CH₂),35.1 (CH), 89.7 (CH), 40.0 (C_(q)), 44.2 (CH), 54.6 (CH), 64.5 (CH₂).

EXAMPLE 30(1S,2S,3S,5S)-(2-Amino-6,6-dimethylbicyclo[3.1.1]hept-3-yl)-methanolhydrochloride (compound 40) (Scheme 8)

The synthesis of 1S,2S,3S,5S enantiomer 40 was accomplished by analogywith Example 29, starting from 1S,2S,3S,5S enantiomer amino ester 31which was prepared according to Example 27; [α]_(D) ²⁰=+8.1 (c=0.5,MeOH); all the spectroscopic data and mp were similar to those for1R,2R,3R,5R enantiomer 28.

EXAMPLE 31(1R,2R,3R,5R)-2-(9H-Fluoren-9-yl-methoxycarbonylamino)-6,6-dimethyl-bicyclo[3.1.1]heptan-3-carboxylicacid (compound 30) (Scheme 6)

The synthesis of 1R,2R,3R,5R enantiomer 30 was accomplished by analogywith Example 13, starting from 1R,2R,3R,5R enantiomer amino acidhydrochloride 8 which was prepared according to Example 26b; mp:155-157° C.; [α]_(D) ²⁰−4 (c=0.25, MeOH); IR=3340, 2926, 1693, 1536,1450, 1252, 739 cm⁻¹. Anal. Calcd. for C₂₅H₂₇NO₄ (405.49): C, 74.05; H,6.71; N, 3.45. Found: C, 74.27; H, 6.52; N, 3.49. ¹H NMR (CDCl₃) δ(ppm): 0.90 (3H, s), 1.20 (3H, s), 1.34 (1H, d, J=10.6 Hz), 1.81-2.14(5H, m), 2.56-2.62 (1H, m), 4.17 (1H, t, J=6.5 Hz), 4.28-4.46 (3H, m),4.97 (1H, br s), 7.20-7.80 (8H, m). ¹³C NMR (CDCl₃) δ (ppm): 20.1, 24.2,27.2, 28.4, 32.1, 40.2, 42.6, 46.7, 47.9, 52.5, 67.5, 120.6, 125.8,127.7, 128.3, 142.0, 144.6, 148.5, 179.8.

EXAMPLE 32(1S,2S,3S,5S)-2-(9H-Fluoren-9-yl-methoxycarbonylamino)-6,6-dimethyl-bicyclo[3.1.1]heptan-3-carboxylicacid (compound 41) (Scheme 8)

The synthesis of 1S,2S,3S,5S enantiomer 41 was accomplished by analogywith Example 13, starting from 1S,2S,3S,5S enantiomer amino acidhydrochloride 9 which was prepared according to Example 28; [α]_(D)²⁰=+6 (c=0.25, MeOH); all the spectroscopic data and mp were similar tothose for 1R,2R,3R,5R enantiomer 30.

EXAMPLE 33 Ethyl(2S,1′R,2′R,3′R,5′R)-2-{[(2′tert-Butoxycarbonylamino)-6′,6′-dimethylbicyclo[3.1.1]heptan-3′-carbonyl)]amino}-3-phenylpropionate(compound 32) (Scheme 6)

The synthesis of 2S,1′R,2′R,3′R,5′R compound 32 was accomplished byanalogy with Example 11, starting from 0.14 g (0.49 mmol) of 1R,2R,3R,5Renantiomer 31.

Isolated compound: 0.09 g (40%); mp: 185-188° C.; [α]_(D) ²⁰=−15(c=0.25, MeOH);

IR=3270, 2926, 1750, 1684, 1651, 1558, 1196 cm⁻¹. Anal. Calcd. forC₂₆H₃₈N₂O₅ (458.59): C, 68.10; H, 8.35; N, 6.11. Found: C, 68.51; H,7.96; N, 6.47. ¹H NMR (CDCl₃) δ (ppm): 0.79 (3H, s), 1.19 (3H, t, J=7.1Hz), 1.20 (3H, s), 1.30 (1H, d, J=10.5 Hz), 1.46 (9H, s), 1.90-2.19 (5H,m), 2.33-2.47 (1H, m), 3.04-3.17 (2H, m), 4.12 (2H, dd, J=14.2, 7.1 Hz),4.20-4.29 (1H, m), 4.25 (1H, t, J=8.6 Hz), 4.77-4.86 (1H, m), 7.17-7.32(5H, m). ¹³C NMR (CDCl₃) δ (ppm): 14.8, 20.1, 24.2, 27.0, 28.2, 29.1,30.4, 38.7, 40.3, 43.6, 47.3, 51.9, 54.5, 61.9, 80.7, 127.5, 129.1,130.1, 137.4, 156.2, 172.6, 174,4.

EXAMPLE 34 Ethyl (2S,1′S,2′S,3′S,5′S)-2-{[(240-tert-Butoxycarbonylamino)-6′,6′-dimethylbicyclo[3.1.1]heptan-340-carbonyl)]amino}-3-phenylpropionate (compound 43) (Scheme 8)

The synthesis of 2S,1′S,2′S,3′S,5′S compound 43 was accomplished byanalogy with Example 11, starting from 0.14 g (0.49 mmol) of 1S,2S,3S,5Senantiomer 42, which was prepared according to Example 28.

Isolated compound: 0.10 g (45%); mp: 186-188° C.; [α]_(D) ²⁰+18 (c=0.25,MeOH); IR=3310, 2926, 1692, 1645, 1557, 1182 cm⁻¹. Anal. Calcd. forC₂₆H₃₈N₂O₅ (458.59): C, 68.10; H, 8.35; N, 6.11. Found: C, 68.39; H,8.05; N, 6.28. ¹H NMR (CDCl₃) δ (ppm): 0.93 (3H, s), 1.21 (3H, t, J=7.1Hz), 1.22 (3H, s), 1.26 (9H, s), 1.31 (1H, d, J=11.1 Hz), 1.94-2.18 (5H,m), 2.29 (1H, m), 3.14 (2H, ddd, J=45.3, 14.1, 6.0 Hz), 4.13 (2H, dd,J=14.1, 7.1 Hz), 4.23-4.32 (1H, m), 4.66 (1H, br s, NH), 4.81-4.91 (1H,m), 6.89 (1H, br s, NH), 7.09-7.30 (5H, m). ¹³C NMR (CDCl₃) δ (ppm):14.8, 20.1, 23.4, 27.2, 29.0, 29.4, 30.0, 32.6, 38.5, 40.3, 43.7, 47.2,54.3, 62.0, 80.3, 127.5, 129.0, 130.0, 137.0, 156.0, 172.6, 174.1.

EXAMPLE 35 (1R,2R,3S,5R)-1-(3-Ethoxycarbonyl-6,6-dimethylbicyclo[3.1.1]heptan-2-yl)-3-(3-methoxyphenyl)-thiourea (compound 44) (Scheme 9)

0.086 g (0.52 mmol) of 3-methoxyphenyl isothiocyanate was added to asolution of 0.100 g (0.47 mmol) of ethyl(1R,2R,3S,5R)-2-amino-6,6-dimethylbicyclo[3.1.1]heptan-3-carboxylate 15(prepared according to Example 7) in 10 ml of toluene. After stirringfor 12 h at room temperature (the reaction was monitored by means ofTLC), the solution was evaporated and crude product was crystallized andwashed with n-hexane.

Isolated compound: 0.154 g (87%); mp: 132-135° C.; [α]_(D) ²⁰+10(c=0.25, MeOH); IR=3315, 3196, 2914, 1711, 1518, 1192, 702 cm⁻¹. Anal.Calcd. for C₂₀H₂₈N₂O₃S (376.51): C, 63.80; H, 7.50; N, 7.44. Found: C,63.61; H, 7.75; N, 7.32. ¹H NMR (CDCl₃) δ (ppm): 0.95 (3H, s), 1.23 (3H,s), 1.23 (3H, t, J=7.1 Hz), 1.47 (1H, d, J=11.1 Hz), 1.91-2.21 (5H, m),3.45 (1H, dt, J=11.1, 4.0 Hz), 3.82 (3H, s, OMe), 4.02-4.08 (2H, ddd,J=14.1, 7.0, 4.0 Hz, CH₂-CH₃), 5.31 (1H, t, J=10.1 Hz), 6.72 (1H, br s),6.73 (1H, d, J=7.0 Hz), 6.81 (1H, d, J=7.1 Hz), 7.31 (1H, t, J=8.1 Hz),7.78 (1H, br s). ¹³C NMR (CDCl₃) δ (ppm): 14.8, 20.9, 25.6, 26.8, 30.0,38.0, 40.0, 40.2, 46.2, 54.6, 56.1, 61.6, 110.6, 112.7, 113.6, 117.1,131.5, 137.1, 160.5 176.2.

EXAMPLE 36 (1S,2S,3R,5S)-1-(3-Ethoxycarbonyl-6,6-dimethylbicyclo[3.1.1]heptan-2-yl)-3-(3-methoxyphenyl)-thiourea (compound 54) (Scheme 11)

The synthesis of 1S,2S,3R,5S enantiomer 54 was accomplished by analogywith Example 35, starting from 1S,2S,3R,5S enantiomer amino ester 34,which was prepared according to Example 8; all the spectroscopic dataand mp were similar to those for 1R,2R,3S,5R enantiomer 44.

Isolated compound: 0.154 g (87%); mp: 133-134° C.; [α]_(D) ²⁰=−6(c=0.25, MeOH).

EXAMPLE 37(1R,2R,3S,5R)-1-(3-Ethoxycarbonyl-6,6-dimethylbicyclo[3.1.1]heptan-2-yl)-3-(4-chlorophenyl)-urea(compound 45) (Scheme 9)

The synthesis of urea derivative 45 was accomplished by analogy withExample 35, starting from 0.100 g (0.47 mmol) of ethyl(1R,2R,3S,5R)-2-amino-6,6-dimethylbicyclo[3.1.1]heptan-3-carboxylate 15(prepared according to Example 7) and 0.079 g (0.52 mmol) of4-chlorophenyl isocyanate.

Isolated compound: 0.153 g (89%); mp: 170-171° C.; [α]_(D) ²⁰=+14(c=0.25, MeOH); IR=3318, 2948, 1725, 1654, 1562, 1493, 1174 cm⁻¹. Anal.Calcd. for C₁₉H₂₅ClN₂O₃ (364.87): C, 62.54; H, 6.91; N, 7.68. Found: C,62.35; H, 7.28; N, 7.43. ¹H NMR (CDCl₃) δ (ppm): 0.93 (3H, s), 1.20 (3H,t, J=7.1 Hz), 1.23 (3H, s), 1.49 (1H, d, J=10.1 Hz), 1.92-2.21 (5H, m),3.31-3.47 (1H, m), 4.08 (2H, dd, J=14.1, 7.1 Hz), 4.73 (1H, t, J=9.1Hz), 5.65 (1H, br s), 7.09 (1H, br s), 7.25 (4H, dd, J=18.1, 9.1 Hz).¹³C NMR (CDCl₃) δ (ppm): 14.8, 21.0, 25.9, 26.9, 29.5, 39.4, 39.5, 40.1,47.4, 48.8, 61.7, 121.6, 128.7, 129.8, 138.4, 155.2, 178.0.

EXAMPLE 38 (1R,2R,3S,5R)-1-(3-Ethoxycarbonyl-6,6-dimethylbicyclo[3.1.1]heptan-2-yl)-3-(3,4-dimethoxyphenylethyl)-thiourea (compound 46) (Scheme9)

The synthesis of thiourea derivative 46 was accomplished by analogy withExample 35, starting from 0.10 g (0.47 mmol) of ethyl(1R,2R,3S,5R)-2-amino-6,6-dimethylbicyclo[3.1.1]heptan-3-carboxylate 15(prepared according to Example 7) and 0.085 g (0.52 mmol) of3,4-dimethoxyphenylethyl isothiocyanate.

Isolated compound: 0.125 g (61%); mp: 125-126° C.; [α]_(D) ²⁰=+20(c=0.25, MeOH); IR=3357, 2936, 1723, 1514, 1260, 1156, 1027, 800 cm⁻¹.Anal. Calcd. for C₂₃H₃₄N₂O₄S (434.59): C, 63.56; H, 7.89; N, 6.45.Found: C, 63.77; H, 7.59; N, 6.61. ¹H NMR (CDCl₃) δ (ppm): 0.93 (3H, s),1.24 (3H, s), 1.24 (3H, t, J=7.1 Hz), 1.55 (1H, d, H-4, J=10.4 Hz),1.95-2.20 (5H, m), 2.80 (2H, t, J=6.9 Hz), 3.36 (1H, dt, J=6.9, 2.6 Hz),3.46-3.61 (2H, m), 3.86 (3H, s), 3.88 (3H, s), 4.00-4.14 (2H, m), 5.12(1H, br s), 5.91 (1H, br s), 6.59 (1H, br s), 6.74-6.82 (3H, m). ¹³C NMR(CDCl₃) δ (ppm): 14.8, 20.9, 25.5, 26.8, 30.0, 35.5, 38.0, 39.9, 40.1,45.6, 46.5, 53.5, 56.6, 61.7, 112.2, 112.6, 121.4, 130.9, 148.6, 149.9,151.9, 177.5.

EXAMPLE 39(1R,2R,3S,5R)-1-Benzyl-1-(3-ethoxycarbonyl-6,6-dimethylbicyclo[3.1.1]heptan-2-yl)-3-(3-methoxyphenyl)-thiourea (compound 47) (Scheme 9)

The synthesis of thiourea derivative 47 was accomplished by analogy withExample 35, starting from 0.142 g (0.47 mmol) of 1R,2R,3S,5R N-benzylamino ester 20 (prepared according to Example 16) and 0.086 g (0.52mmol) of 3-methoxyphenyl isothiocyanate.

Isolated compound: 0.148 g (68%); mp: 109-112° C.; [α]_(D) ²⁰=−4(c=0.25,MeOH); IR=3346, 2898, 1713, 1597, 1493, 1186, 1032, 850 cm⁻¹. Anal.Calcd. for C₂₇H₃₄N₂O₃S (466.64): C, 69.50; H, 7.34; N, 6.00. Found: C,69.61; H, 7.11; N, 6.32. ¹H NMR (CDCl₃) δ (ppm): 0.99 (3H, s), 1.23 (3H,s), 1.31 (3H, t, J=7.1 Hz), 1.91-2.11 (4H, m), 2.22 (1H, d, H-4, J=11.1Hz), 2.27-2.37 (1H, m), 3.72 (3H, s), 4.02 (1H, t, J=7.1 Hz), 4.17 (2H,dd, J=14.1, 7.1 Hz), 4.53-4.68 (1H, m), 4.93 (1H, d, J=18.1 Hz), 6.03(1H, br s), 6.54 (1H, d, J=8.1 Hz), 6.61-6.73 (2H, m), 6.88 (1H, br s,NH), 7.11 (1H, t, J=7.1 Hz), 7.22-7.46 (5H, m). ¹³C NMR (CDCl₃) δ (ppm):15.1, 21.2, 26.9, 27.2, 29.1, 38.1, 39.4, 41.5 44.5, 51.2, 55.9, 60.6,61.6, 111.9, 118.3, 125.4, 126.5, 128.3, 128.7, 129.8, 130.0, 136.1,140.5, 160.2, 176.7.

EXAMPLE 40(1R,2R,3S,5R)-1-Benzyl-1-(3-ethoxycarbonyl-6,6-dimethylbicyclo[3.1.1]heptan-2-yl)-3-(4-chlorophenyl)-urea(compound 48) (Scheme 9)

The synthesis of urea derivative 48 was accomplished by analogy withExample 35, starting from 0.142 g (0.47 mmol) of 1R,2R,3S,5R N-benzylamino ester 20 (prepared according to Example 16) and 0.079 g (0.52mmol) of 4-chlorophenyl isocyanate.

Isolated compound: 0.161 g (75%); mp: 202-205° C.; [α]_(D) ²⁰=+5(c=0.25, MeOH); IR=3321, 2940, 1723, 1636, 1526, 1493, 1241, 1178, 828,696 cm⁻¹. Anal. Calcd. for C₂₆H₃₁ClN₂O₃ (454.99): C, 68.63; H, 6.87; N,6.16. Found: C, 68.35; H, 6.96; N, 5.93. ¹H NMR (CDCl₃) δ (ppm): 0.94(3H, s), 1.19 (3H, t, J=7.1 Hz), 1.23 (3H, s), 1.90-2.18 (4H, m, 2.21(1H, d, J=10.1 Hz), 2.27-2.33 (1H, m), 3.65 (1H, dt, J=10.1, 3.0 Hz),4.10 (2H, ddd, J=14.1, 7.1, 2.0 Hz), 4.31 (1H, d, J=18.1 Hz), 4.64 (1H,d, J=18.1 Hz), 5.10 (1H, d, J=9.1 Hz), 6.11 (1H, br s, NH), 7.00 (2H, d,J=8.1 Hz), 7.12 (2H, d, J=8.1 Hz), 7.22-7.42 (5H, m). ¹³C NMR (CDCl₃) δ(ppm): 14.9, 20.9, 26.8, 27.1, 29.0, 39.3, 39.5, 41.2, 44.3, 48.6, 55.6,61.7, 121.5, 126.7, 128.5, 128.6, 129.3, 130.0, 138.3, 138.6, 156.1,177.6.

EXAMPLE 41 (1R,2R,3R,5R)-1 -(3-Ethoxycarbonyl-6,6-dimethylbicyclo[3.1.1]heptan-2-yl)-3-(3-methoxyphenyl)-thiourea (compound 49) (Scheme 10)

The synthesis of thiourea derivative 49 was accomplished by analogy withExample 35, starting from 0.10 g (0.47 mmol) of ethyl(1R,2R,3R,5R)-2-amino-6,6-dimethylbicyclo[3.1.1]heptan-3-carboxylate 26(prepared according to Example 23) and 0.086 g (0.52 mmol) of3-methoxyphenyl isothiocyanate.

Isolated compound: 0.145 g (82%); mp: 118-120° C.; [α]_(D) ²⁰=−86(c=0.25, MeOH); IR=3344, 3158, 2918, 1731, 1540, 1148, 694 cm⁻¹. Anal.Calcd. for C₂₀H₂₈N₂O₃S (376.51): C, 63.80; H, 7.50; N, 7.44. Found: C,63.69; H, 7.63; N, 7.21. ¹H NMR (CDCl₃) δ (ppm): 0.96 (3H, s), 1.25 (3H,s), 1.30 (3H, t, J=7.1 Hz), 1.47 (1H, br s), 1.91-2.23 (5H, m), 2.6 (1H,br s), 3.81 (3H, s), 4.23 (2H, dd, J=14.1, 7.0), 5.11 (1H, br s), 6.25(1H, br s), 6.72-7.01 (3H, m), 7.30 (1H, t, J=8.1 Hz), 7.81 (1H, br s).¹³C NMR (CDCl₃) δ (ppm): 14.9, 20.1, 24.0, 27.0, 27.9, 29.2, 40.1, 42.1,46.3, 56.1, 62.1, 110.5, 112.5, 116.9, 130.5, 160.8, 180.3.

EXAMPLE 42(1S,2S,3S,5S)-1-(3-Ethoxycarbonyl-6,6-dimethylbicyclo[3.1.1]heptan-2-yl)-3-(3-methoxyphenyl)-thiourea(compound 55) (Scheme 12)

The synthesis of 1S,2S,3S,5S enantiomer 55 was accomplished by analogywith Example 35, starting from 0.100 g (0.47 mmol) of 1S,2S,3S,5Senantiomer amino ester 39, which was prepared according to Example 24;all the spectroscopic data and mp were similar to those for 1R,2R,3R,5Renantiomer 49.

Isolated compound: 0.145 g (82%); mp: 120-122° C.; [α]_(D) ²⁰=+22(c=0.25, MeOH).

EXAMPLE 43(1R,2R,3R,5R)-1-(3-Ethoxycarbonyl-6,6-dimethylbicyclo[3.1.1]heptan-2-yl)-3-(4-chlorophenyl)-urea(compound 50) (Scheme 10)

The synthesis of urea derivative 48 was accomplished by analogy withExample 35, starting from 0.10 g (0.47 mmol) of ethyl(1R,2R,3R,5R)-2-amino-6,6-dimethylbicyclo[3.1.1]heptan-3-carboxylate 26(prepared according to Example 23) and 0.079 g (0.52 mmol) of4-chlorophenyl,isocyanate.

Isolated compound: 0.133 g (78%); mp: 167-169° C.; [α]_(D) ²⁰=−38(c=0.25, MeOH); IR=3328, 2926, 1727, 1654, 1558, 1493, 1156, 830cm^(−1.) Anal. Calcd. for C₁₉H₂₅ClN₂O₃ (364.87): C, 62.54; H, 6.91; N,7.68. Found: C, 62.75; H, 6.78; N, 7.53. ¹H NMR (CDCl₃) δ (ppm): 0.90(3H, s), 1.23 (3H, s), 1.27 (3H, t, J=7.1 Hz), 1.37 (1H, d, H-4, J=10.1Hz), 1.86-2.08 (3H, m), 2.13-2.28 (2H, m), 2.64-2.74 (1H, m), 4.21 (2H,dd, J=14.1, 7.1 Hz), 4.37 (1H, d, J=8.1 Hz), 7.20 (1H, d, J=8.1 Hz),7.32 (1H, d, J=8.1 Hz), 7.91 (1H, br s, NH). ¹³C NMR (CDCl₃) δ (ppm):14.8, 20.2, 24.1, 27.0, 28.7, 40.2, 40.5, 43.4, 47.2, 52.1, 62.1, 121.1,127.9, 129.5, 137.7, 155.3, 176.8.

EXAMPLE 44 (1S,2S,3S,5S)-1-(3-Ethoxycarbonyl-6,6-dimethylbicyclo[3.1.1]heptan-2-yl)-3-(4-chlorophenyl)-urea (compound 56) (Scheme 12)

The synthesis of 1S,2S,3S,5S enantiomer 56 was accomplished by analogywith Example 35, starting from 0.10 g (0.47 mmol) of ethyl1S,2S,3S,55-2-amino-6,6-dimethylbicyclo[3.1.1]heptan-3-carboxylateenantiomer amino ester 39, which was prepared according to Example 24,and 0.079 g (0.52 mmol) of 4-chlorophenyl isocyanate; all thespectroscopic data and mp were similar to those for 1R,2R,3R,5Renantiomer 50.

Isolated compound: 0.125 g (73%); mp: 167-169° C.; [α]_(D) ²⁰=+16(c=0.25, MeOH).

EXAMPLE 45(1R,2R,3R,5R)-1-(3-Ethoxycarbonyl-6,6-dimethylbicyclo[3.1.1]heptan-2-yl)-3-(3,4-dimethoxyphenyl-ethyl)-thiourea(compound 51) (Scheme 10)

The synthesis of thiourea derivative 51 was accomplished by analogy withExample 35, starting from 0.10 g (0.47 mmol) of ethyl(1R,2R,3R,5R)-2-amino-6,6-dimethylbicyclo[3.1.1]heptan-3-carboxylate 26(prepared according to Example 23) and 0.085 g (0.52 mmol) of3,4-dimethoxyphenylethyl isothiocyanate with that modification, that theoily crude product was purified by flash chromatography on a silica gelcolumn (toluene:ethanol=9:1) resulting in compound 51.

Isolated compound: 0.120 g (59%); oil; [α]_(D) ²⁰=−32 (c=0.25, MeOH);IR=3350, 2932, 1729, 1514, 1261, 1025, 806 cm⁻¹. Anal. Calcd. forC₂₃H₃₄N₂O₄S (434.59): C, 63.56; H, 7.89; N, 6.45. Found: C, 63.65; H,7.61; N, 6.73. ¹H NMR (CDCl₃) δ (ppm): 0.78 (3H, s), 1.22 (3H, s), 1.26(3H, t, J=7.1 Hz), 1.37 (1H, d, J=11.1 Hz), 1.77 (1H, dd, J=10.1, 13.0),1.96-2.01 (2H, m), 2.16-2.34 (2H, m), 2.67-2.97 (3H, m), 3.76-3.87 (8H,m), 4.10-4.25 (3H, m), 6.05 (1H, d, J=9.1 Hz), 6.78-6.83 (3H, m), 7.10(1H, br s, NH). ¹³C NMR (CDCl₃) δ (ppm): 14.8, 20.2, 24.4, 26.8, 29.1,35.3, 40.1, 40.2, 42.4, 46.6, 47.6, 53.9, 56.5, 56.6, 62.2, 112.0,112.8, 121.4, 128.8, 132.1, 137.6, 148.7, 176,7.

EXAMPLE 46(1R,2R,3R,5R)-1-Benzyl-1-(3-Ethoxycarbonyl-6,6-dimethylbicyclo[3.1.1]-heptan-2-yl)-3-(3-methoxyphenyl)-thiourea(compound 52) (Scheme 10)

The synthesis of thiourea derivative 52 was accomplished by analogy withExample 35, starting from 0.142 g (0.47 mmol) of 1R,2R,3R,5R N-benzylamino ester 27 (prepared according to Example 25) and 0.086 g (0.52mmol) of 3-methoxyphenyl isothiocyanate.

Isolated compound: 0.140 g (64%); mp: 110-111° C.; [α]_(D) ²⁰=+2(c=0.25, MeOH); IR=3340, 2936, 1710, 1610, 1543, 1496, 1282, 1057 cm⁻¹.Anal. Calcd. for C₂₇H₃₄N₂O₃S (466.64): C, 69.50; H, 7.34; N, 6.00.Found: C, 69.41; H, 7.53; N, 6.12. ¹H NMR (CDCl₃) δ (ppm): 0.90 (3H, s),1.26 (3H, s), 1.27 (3H, t, J=7.1 Hz), 1.61 (1H, d, J=10.7 Hz), 1.76 (1H,dd, J=13.2, 10.1 Hz), 1.90-1.93 (1H, m), 2.09-2.16 (2H, m), 2.34-2.39(1H, m), 2.86 (1H, dd, J=19.7, 9.8 Hz), 3.82 (3H, s), 4.13-4.30 (2H, m),4.57 (1H, d, J=16.8 Hz), 5.37 (1H, d, J=9.7 Hz), 6.69 (1H, dd, J=10.3,2.1 Hz), 7.06 (1H, d, J=7.8 Hz), 7.21-7.31 (7H, m), 9.52 (1H, br s). ¹³CNMR (CDCl₃) δ (ppm): 14.8, 20.2, 26.0, 27.2, 29.0, 39.1, 39.4, 42.0,46.6, 50.0, 56.0, 59.1, 62.4, 110.7, 111.0, 117.1, 126.8, 127.6, 129.2,129.7, 142.6, 185.4.

EXAMPLE 47(1R,2R,3R,5R)-1-Benzyl-1-(3-Ethoxycarbonyl-6,6-dimethylbicyclo[3.1.1]-heptan-2-yl)-3-(4-chlorophenyl)-urea(compound 53) (Scheme 10)

The synthesis of urea derivative 53 was accomplished by analogy withExample 35, starting from 0.142 g (0.47 mmol) of 1R,2R,3R,5R N-benzylamino ester 27 (prepared according to Example 25) and 0.079 g (0.52mmol) of 4-chlorophenyl isocyanate with that modification, that the oilycrude product was purified by flash chromatography on a silica gelcolumn (toluene:ethanol=9:1) resulting in compound 53.

Isolated compound: 0.150 g (70%); oil; [α]_(D) ²⁰=+4 (c=0.25, MeOH);IR=3332, 2926, 1704, 1676, 1595, 1496, 1211, 830 cm⁻¹. Anal. Calcd. forC₂₆H₃₁ClN₂O₃ (454.99): C, 68.63; H, 6.87; N, 6.16. Found: C, 68.75; H,6.63; N, 6.41. ¹H NMR (CDCl₃) δ (ppm): 0.92 (3H, s), 1.23 (3H, s), 1.26(3H, t, J=7.1 Hz), 1.57 (1H, d, J=10.7 Hz), 1.76 (1H, dd, J=13.2, 10.1Hz), 1.89-1.93 (1H, m), 2.02 (1H, t, J=5.3 Hz), 2.11-2.18 (1H, m),2.28-2.35 (1H, m), 2.86 (1H, dd, J=19.5, 9.7 Hz), 4.14-4.30 (3H, m),4.80 (1H, d, J=9.7 Hz), 4.87 (1H, d, J=16.6 Hz), 7.17-7.39 (9H, m), 8.53(1H, br s). ¹³C NMR (CDCl₃) δ (ppm): 14.9, 20.2, 26.0, 27.2, 29.0, 39.4,39.7, 41.7, 45.7, 46.3, 56.2, 62.2, 121.3, 127.0, 127.6, 127.7, 129.2,129.3, 139.4, 140.3, 157.0, 177.9.

In the enclosed FIG. 2 relating to the Summarizing Formula Table themeanings of substituents X and Y are given by reference to theindividual Examples and to the numbers of the compounds preparedaccording to the Examples.

Biological Assays

The following examinations carried out in connection with the multidrugresistance reversing effect of the compounds according to the inventionprove that the novel compounds are modulators of the mdr1 genetransfected into the mouse lymphoma cell line.

Cell Cultures

L5178 mouse T-cell lymphoma cells were transfected with pHa MDR1/Aretrovirus, by the method as described by J. L. Weaver [J. L. Weaver, G.Szabó, P. S. Pine, M. M. Gottesmann, S. Goldenberg and A. Aszalos: Theeffect of ion channel blockers, immunosuppressive agents and other drugson the activity of the multidrug transporter. Int. J. Cancer 54, 456-61(1993)]. Mdr-1-expressing cell lines were selected by culturing thetransfected cells with 60 ng/mL of colchicine to maintain the expressionof the MDR phenotype. L5178 (parent) mouse T-cell lymphoma cells and thehuman mdr1 transfected subline were cultured in McCoy's 5A mediumsuplemented with 10% by mass of health-inactivated horce serum,L-glutamine and antibiotics. Both cell lines were cultured at 37° C. Themouse lymphoma cell line was maintained in a 5% CO₂ atmosphere.

Method

Reversal of Multidrug Resistance

As pre-assay, the concentrations inhibiting 50% of cell proliferationwere determined. Based on the ID₅₀ values indicating theantiproliferative effect of the tested compounds, they proved to benon-cytotoxic under the test conditions; consequently the measurementscould be performed.

The assays were carried out by the method as described by J. Molnár etal. [J. Molnár, I. Mucsi, J. Nacsa, A. Hevér, N. Gyémánt, K. Ugocsai, P.Hegyes, S. Kiessig, D. Gaal, H. Lage and A. Varga: New silicon compoundsas resistance modifiers against multidrug-resistant cells. AnticancerResearch 24, 865-71 (2004)]. The cell density was set to a value of2×10⁶/mL, then the cells were resuspended in a serum-free McCoy's 5Amedium and divided in aliquots of 0.5 mL in Eppendorf centrifuge tubes.The tested compounds were added in different concentrations to differentvolumes (2.0-20.0 μL) of 1.0-10.0 mg/mL stock solutions and the sampleswere incubated for 10 minutes at room temperature. Thereafter 10 μL (5.2μM final concentration) of indicator rhodamine 123 were added to eachsample [D. Kessel: Exploring multidrug resistance using rhodamine 1223,Cancer Commun. 145-149 (1989)] and the cells were incubated for further20 minutes at 37° C., then washed twice with phosphate buffer andresuspended in 0.5 ml of phosphate buffer for analysis. The fluorescenceof the cell population was measured with a Beckton Dickinson FACScanflow cytometer. Verapamil was used as a positive control in therhodamine 123 experiments [M. M. Cornwell, I. Pastan and M. M.Gottesmann: Certain calcium channel blockers bind specifically tomultidrug resistant human KB carcinoma membrane vesicles and inhibitdrug binding t P-glycoprotein. J. Biol. Chem. 262, 2166-70 (1987)]. Thepercentage value of the mean fluorescence intensity was calculated forthe treated MDR and for the parental cell lines as compared with thevalues obtained with the untreated cells. The activity ratio (R) wascalculated on the basis of the measured fluorescence values by thefollowing equation:

$R = \frac{{MDR}\mspace{14mu} {{treated}/{MDR}}\mspace{14mu} {control}}{{parental}\mspace{14mu} {{treated}/{parental}}\mspace{14mu} {control}}$

Results

Multidrug Resistance Reversing Effect

In our assays the compounds were tested in two concentrations (4 and 40μg/mL), wherein Verapamil was used as positive in vitro control (Tables2 and 3). A significant dose-dependent effect was observed with thecompounds of formulas 44, 46, 47, 49, 50, 51 and 56: in higherconcentrations they gave multiple values of the fluorescence activity ofthe positive control Verapamil measured at the concentration used.

Compounds of formulas 47 and 52 showed even at lower concentrations aconsiderable multidrug resistance reversing effect; at higherconcentrations similar fluorescence activity values were measured (Table3), indicating saturation.

The effect of compound of formula 47 was separately tested in aconcentration series (0.04, 0.08, 0.4, 0.8 and 4 μg/mL). Dose-dependencewas observed: it can be noted that already a concentration of 0.4 μg/mLof this compound is sufficient for exerting a significantP-glycoprotein-inhibiting effect (Table 4).

TABLE 2 Number of formula of the compound μg/ml R Vrel Verapamil 10 4.921.00 46 4 4.02 0.82 40 21.92 4.46 51 4 13.29 2.70 40 49.02 9.96 44 42.02 0.41 40 22 4.47 49 4 2.77 0.56 40 28.07 5.71 54 4 2.37 0.48 4026.35 5.36 55 4 3.07 0.62 40 11.99 2.44 45 4 7.46 1.52 40 10.89 2.21 384 2.33 0.79 40 19.92 6.73

TABLE 3 Number of formula of the compound μg/ml R Vrel Verapamil 1011.45 1.00 50 4 1.87 0.16 40 42.58 3.70 56 4 1.57 0.14 40 39.39 3.43 474 34.76 3.02 40 35.35 3.07 52 4 21.87 1.90 40 24.44 2.13 41 4 0.86 0.0740 1.25 0.11 17 4 23.1 1.58 40 94.24 6.45 R = ratio of fluorescence andactivity Vrel = effect related to Verapamil

TABLE 4 Tested compound c (μg/ml) R Verapamil 10 21.37 Compound offormula 47 4 35.20 0.8 7.16 0.4 6.03 0.08 1.60 0.04 1.44

1-10. (canceled)
 11. Chiral cyclic2-amino-6,6-dimethylbicyclo[3.1.1]heptane-3-carboxylic acid derivativesof general formula (I)—

wherein R stands for C₁₋₄ Alk; X stands for —COOH, —CONH₂, —CONH (C₁₋₄Alk), —CON(C₁₋₄ Alk)₂, —COO(C₁₋₄ Alk), —COPhe-O-(C₁₋₄ Alk) or —CH₂OH; Ystands for —NH₂, —NHBoc, —NHFmoc, —NH(C₁₋₄ Alk), —N (C₁₋₄ Alk)₂,—NHCH₂Ph, or Ar—NH—C(=X⁰)—N(R⁰)— wherein Ar stands for a phenyl groupsubstituted by one or two C₁₋₄ alkoxy group(s) or by one halogen, X⁰stands for O or S, and R⁰ stands for hydrogen or benzyl; and X+Y standfor —CONH— or —CON(Boc)-; with the proviso that when X stands for —COOH,then Y may be only different from —NH₂— and their salts formed withpharmaceutically acceptable acids or bases.
 12. A compound as claimed inclaim 11 selected from the group consisting of(1R,2R,5S,7R)-N-tert-butoxycarbonyl-8,8-dimethyl-3-aza-tricyclo[5.1.1.0^(2,5)]nonane-4-one(compound of formula 11),(1R,2R,3R,5R)-2-(9H-fluoren-9-yl-methoxycarbonylamino)-6,6-dimethyl-bicyclo[3.1.1]heptane-3-carboxylicacid (compound of formula 30),(1R,2R,3S,5R)-1-(3-ethoxycarbonyl-6,6-dimethyl-bicyclo[3.1.1]heptane-2-yl)-3-(3-methoxyphenyl)-thiourea(compound of formula 44),(1R,2R,3S,5R)-1-(3-ethoxycarbonyl-6,6-dimethyl-bicyclo[3.1.1]heptane-2-yl)-3-(3,4-dimethoxy-phenylethyl)-thiourea(compound of formula 46),(1R,2R,3S,5R)-1-benzyl-1-(3-ethoxycarbonyl-6,6-dimethylbicyclo[3.1.1]heptane-2-yl)-3-(3-methoxyphenyl)-thiourea(compound of formula 47),(1R,2R,3R,5R)-1-(3-ethoxycarbonyl-6,6-dimethyl-bicyclo[3.1.1]heptane-2-yl)-3-(3-methoxyphenyl)-thiourea(compound of formula 49),(1R,2R,3R,5R)-1-(3-ethoxycarbonyl-6,6-dimethyl-bicyclo[3.1.1]heptane-2-yl)-3-(4-chlorophenyl)-urea(compound of formula 50),(1R,2R,3R,5R)-1-(3-ethoxycarbonyl-6,6-dimethyl-bicyclo[3.1.1]heptane-2-yl)-3-(3,4-dimethoxyphenyl-ethyl)-thiourea(compound of formula 51),(1R,2R,3R,5R)-1-benzyl-1-(3-ethoxycarbonyl-6,6-dimethylbicyclo[3.1.1]heptane-2-yl)-3-(3-methoxyphenyl)-thiourea(compound of formula 52), and(1S,2S,3S,5S)-1-(3-ethoxycarbonil-6,6-dimethyl-bicyclo[3.1.1]heptane-2-yl)-3-(4-chlorophenyl)-urea(compound of formula 56). 13.2-Amino-6,6-dimethyl-bicyclo[3.1.1]heptane-3-carboxylic acids and theirsalts formed with pharmaceutically acceptable acids and bases, resp., asclaimed in claim
 11. 14. A compound as claimed in claim 11 selected fromthe group consisting of(1R,2R,3S,5R)-2-Amino-6,6-dimethyl-bicyclo[3.1.1]heptane-3-carboxylicacid of formula 6,(1S,2S,3R,5S)-2-Amino-6,6-dimethyl-bicyclo[3.1.1]heptane-3-carboxylicacid of formula 7,(1R,2R,3R,5R)-2-Amino-6,6-dimethyl-bicyclo[3.1.1]heptane-3-carboxylicacid of formula 8,(1S,2S,3S,5S)-2-Amino-6,6-dimethyl-bicyclo[3.1.1]heptane-3-carboxylicacid of formula
 9. 15. A pharmaceutical composition reversing multidrugresistance which comprises one or more compounds of general formula (I)as active agent and/or the salts thereof as claimed in claim 11 andusual inert pharmaceutical carriers and/or auxiliary agents.
 16. Amethod for preparing multidrug-resistance reversing pharmaceuticalcompositions comprising mixing one or more compound(s) of generalformula (I) and/or the salts thereof as claimed in claim 11 with aninert pharmaceutical carrier and/or auxiliary agents.
 17. A method forreversing multidrug resistance comprising administering an effectiveamount of the composition of claim 15 to a subject in need of suchtreatment.